Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death.

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric ß-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.

De-ubiquitinating protease USP2a targets RIP1 and TRAF2 to mediate cell death by TNF.

Components of the TNFR1 complex are subject to dynamic ubiquitination that impacts on their effects as signalling factors. We have found that the ubiquitin-specific protease USP2a has a pivotal role in the decision for cell death or survival by the TNFR1 complex. This enzyme is a novel component of the TNFR1 complex that is recruited upon ligand binding and controls the signalling activity of the TNFR1-interacting protein RIP1 by removing its K63-linked ubiquitin chains. USP2a similarly de-ubiquitinates TRAF2, a ubiquitin-ligase recruited to the TNFR1 complex. During the TNF response the activity of USP2a on RIP1 and TRAF2 is required for the efficient reappearance of IκBα, which is essential to inactivate the anti-apoptotic transcription factor NF-κB. The effects of USP2a culminate in the conversion of the anti-apoptotic TNFR1 complex I into the pro-apoptotic TNFR1 complex II. Consequently, downregulation of USP2a promotes NF-κB activation and protects cells against TNF-induced cell death.

Specific disintegration of complex II succinate:ubiquinone oxidoreductase links pH changes to oxidative stress for apoptosis induction.

The formation of reactive oxygen species (ROS) and the change of the intracellular pH (pH(i)) are common phenomena during apoptosis. How they are interconnected, however, is poorly understood. Here we show that numerous anticancer drugs and cytokines such as Fas ligand and tumour necrosis factor α provoke intracellular acidification and cause the formation of mitochondrial ROS. In parallel, we found that the succinate:ubiquinone oxidoreductase (SQR) activity of the mitochondrial respiratory complex II is specifically impaired without affecting the second enzymatic activity of this complex as a succinate dehydrogenase (SDH). Only in this configuration is complex II an apoptosis mediator and generates superoxides for cell death. This is achieved by the pH(i) decline that leads to the specific dissociation of the SDHA/SDHB subunits, which encompass the SDH activity, from the membrane-bound components of complex II that are required for the SQR activity.

Isolation of ORCTL3 in a novel genetic screen for tumor-specific apoptosis inducers.

We have established a systematic high-throughput screen for genes that cause cell death specifically in transformed tumor cells. In a first round of screening, cDNAs that induce apoptosis in a transformed human cell line are detected. Positive genes are subsequently tested in a synthetic lethal screen in normal cells versus their isogenic counterparts that have been transformed by a particular oncogene. In this way, the organic cation transporter-like 3 (ORCTL3) gene was found to be inactive in normal rat kidney (NRK) cells, but to induce apoptosis in NRK cells transformed by oncogenic H-ras. ORCTL3 also causes cell death in v-src-transformed cells and in various human tumor cell lines but not in normal cells or untransformed cell lines. Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity. Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism. Finally, we detected ORCTL3 to be downregulated in human kidney tumors.