Francesca Macchi

Endonuclease G mediates α‐synuclein cytotoxicity during Parkinson's disease

Malfunctioning of the protein α‐synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinsons disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson‐diseased patients, while EndoG depletion largely reduces α‐synuclein‐induced cell death in human neuroblastoma cells. Xenogenic expression of human α‐synuclein in yeast cells triggers mitochondria‐nuclear translocation of EndoG and EndoG‐mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α‐synuclein‐driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α‐synuclein‐expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α‐synuclein cytotoxicity.

Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors

Testing of new therapeutic strategies for Parkinsons disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process.

FK506 reduces neuroinflammation and dopaminergic neurodegeneration in an α-synuclein-based rat model for Parkinson's disease.

Alpha-synuclein (α-synuclein) is considered a key player in Parkinsons disease (PD), but the exact relationship between α-synuclein aggregation and dopaminergic neurodegeneration remains unresolved. There is increasing evidence that neuroinflammatory processes are closely linked to dopaminergic cell death, but whether the inflammatory process is causally involved in PD or rather reflects secondary consequences of nigrostriatal pathway injury is still under debate. We evaluated the therapeutic effect of the immunophilin ligand FK506 in a rAAV2/7 α-synuclein overexpression rat model. Treatment with FK506 significantly increased the survival of dopaminergic neurons in a dose-dependent manner. No reduction in α-synuclein aggregation was apparent in this time window, but FK506 significantly lowered the infiltration of both T helper and cytotoxic T cells and the number and subtype of microglia and macrophages. These data suggest that the anti-inflammatory properties of FK506 decrease neurodegeneration in this α-synuclein-based PD model, pointing to a causal role of neuroinflammation in the pathogenesis of PD.

Alpha-synuclein-induced neurodegeneration is exacerbated in PINK1 knockout mice

Loss-of-function mutations in the PINK1 gene lead to recessive forms of Parkinsons disease. Animal models with depleted PINK1 expression have failed to reproduce significant nigral dopaminergic neurodegeneration and clear alpha-synuclein pathology, main characteristics of the disease. In this study, we investigated whether alpha-synuclein pathology is altered in the absence of PINK1 in cell culture and in vivo. We observed that downregulation of PINK1 enhanced alpha-synuclein aggregation and apoptosis in a neuronal cell culture model for synucleinopathy. Silencing of PINK1 expression in mouse substantia nigra using recombinant adeno-associated viral vectors did not induce dopaminergic neurodegeneration in a long-term study up to 10 months, nor did it enhance or accelerate dopaminergic neurodegeneration after alpha-synuclein overexpression. However, in PINK1 knockout mice, overexpression of alpha-synuclein in the substantia nigra resulted in enhanced dopaminergic neurodegeneration as well as significantly higher levels of alpha-synuclein phosphorylation at serine 129 at 4 weeks postinjection. In conclusion, our results demonstrate that total loss of PINK1 leads to an increased sensitivity to alpha-synuclein-induced neuropathology and cell death in vivo.

Noninvasive Bioluminescence Imaging of α-Synuclein Oligomerization in Mouse Brain Using Split Firefly Luciferase Reporters

Alpha-synuclein (αSYN) aggregation plays a pivotal role in the pathogenesis of Parkinsons disease and other synucleinopathies. In this multistep process, oligomerization of αSYN monomers is the first step in the formation of fibrils and intracytoplasmic inclusions. Although αSYN oligomers are generally considered to be the culprit of these diseases, the methodology currently available to follow-up oligomerization in cells and in brain is inadequate. We developed a split firefly luciferase complementation system to visualize oligomerization of viral vector-encoded αSYN fusion proteins. αSYN oligomerization resulted in successful luciferase complementation in cell culture and in mouse brain. Oligomerization of αSYN was monitored noninvasively with bioluminescence imaging in the mouse striatum and substantia nigra up to 8 months after injection. Moreover, the visualized αSYN oligomers retained their toxic and aggregation properties in both model systems. Next, the effect of two small molecules, FK506 and (-)-epigallocatechin-3-gallate (EGCG), known to inhibit αSYN fibril formation, was investigated. FK506 inhibited the observed αSYN oligomerization both in cell culture and in mouse brain. In conclusion, the split firefly luciferase-αSYN complementation assay will increase our insight in the role of αSYN oligomers in synucleinopathies and opens new opportunities to evaluate potential αSYN-based neuroprotective therapies.

High-content analysis of α-synuclein aggregation and cell death in a cellular model of Parkinson's disease.

BACKGROUND Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinsons disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies. NEW METHOD Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis. RESULTS We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells. COMPARISON WITH EXISTING METHODS Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner. CONCLUSIONS The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.