Angélique Deleersnijder

Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

α-Synuclein (α-SYN) is a key player in the pathogenesis of Parkinsons disease (PD). In pathological conditions, the protein is present in a fibrillar, aggregated form inside cytoplasmic inclusions called Lewy bodies. Members of the FK506 binding protein (FKBP) family are peptidyl-prolyl isomerases that were shown recently to accelerate the aggregation of α-SYN in vitro. We now established a neuronal cell culture model for synucleinopathy based on oxidative stress-induced α-SYN aggregation and apoptosis. Using high-content analysis, we examined the role of FKBPs in aggregation and apoptotic cell death. FK506, a specific inhibitor of this family of proteins, inhibited α-SYN aggregation and neuronal cell death in this synucleinopathy model dose dependently. Knockdown of FKBP12 or FKBP52 reduced the number of α-SYN aggregates and protected against cell death, whereas overexpression of FKBP12 or FKBP52 accelerated both aggregation of α-SYN and cell death. Thus, FK506 likely targets FKBP members in the cell culture model. Furthermore, oral administration of FK506 after viral vector-mediated overexpression of α-SYN in adult mouse brain significantly reduced α-SYN aggregate formation and neuronal cell death. Our data explain previously described neuroregenerative and neuroprotective effects of immunophilin ligands and validate FKBPs as a novel drug target for the causative treatment 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.

Comparative Analysis of Different Peptidyl-Prolyl Isomerases Reveals FK506-binding Protein 12 as the Most Potent Enhancer of α-Synuclein Aggregation

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75.

Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.

Unraveling the Role of Peptidyl-Prolyl Isomerases in Neurodegeneration

Immunophilins are a family of highly conserved proteins with a peptidyl-prolyl isomerase activity that binds immunosuppressive drugs such as FK506, cyclosporin A, and rapamycin. Immunophilins can be divided into two subfamilies, the cyclophilins, and the FK506 binding proteins (FKBPs). Next to the immunophilins, a third group of peptidyl-prolyl isomerases exist, the parvulins, which do not influence the immune system. The beneficial role of immunophilin ligands in neurodegenerative disease models has been known for more than a decade but remains largely unexplained in terms of molecular mechanisms. In this review, we summarize reported effects of parvulins, immunophilins, and their ligands in the context of neurodegeneration. We focus on the role of FKBP12 in Parkinson’s disease and propose it as a novel drug target for therapy of Parkinson’s disease.

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.