Exosomal cell-to-cell transmission of alpha synuclein oligomers

Background: Aggregation of alpha-synuclein (αsyn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson’s disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of αsyn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted αsyn across membranes. The aim of this study is to characterize the transcellular spread of αsyn oligomers and determine their extracellular location. Results: Using a novel protein fragment complementation assay where αsyn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that αsyn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated αsyn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free αsyn oligomers. Specifically, we determine that αsyn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of αsyn oligomers is strongly influenced by autophagic activity. Conclusions: Our data suggest that αsyn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of αsyn oligomers is a mechanism whereby cells clear toxic αsyn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in αsyn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies

Brain-Permeable Small-Molecule Inhibitors of Hsp90 Prevent α-Synuclein Oligomer Formation and Rescue α-Synuclein-Induced Toxicity

Aggregation of α-synuclein (αsyn) is a hallmark of sporadic and familial Parkinson's disease (PD) and dementia with Lewy bodies. Lewy bodies contain αsyn and several heat shock proteins (Hsp), a family of molecular chaperones up-regulated by the cell under stress. We have previously shown that direct expression of Hsp70 and pharmacological up-regulation of Hsp70 by geldanamycin, an Hsp90 inhibitor, are protective against αsyn-induced toxicity and prevent aggregation in culture. Here, we use a novel protein complementation assay to screen a series of small-molecule Hsp90 inhibitors for their ability to prevent αsyn oligomerization and rescue toxicity. By use of this assay, we found that several compounds prevented αsyn oligomerization as measured by decreased luciferase activity, led to a reduction in high-molecular-mass oligomeric αsyn, and protected against αsyn cytotoxicity. A lead compound, SNX-0723 (2-fluoro-6-[(3S)-tetrahydrofuran-3-ylamino]-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide) was determined to have an EC50 for inhibition of αsyn oligomerization of approximately 48 nM and was able to rescue αsyn-induced toxicity. In vivo assessment of SNX-0723 showed significant brain concentrations along with induction of brain Hsp70. With a low EC50, brain permeability, and oral availability, these novel inhibitors represent an exciting new therapeutic strategy for PD

Tertiary Lymphoid Structures in Rheumatoid Arthritis: NF-κB-Inducing Kinase-Positive Endothelial Cells as Central Players

Tertiary lymphoid structures (TLSs) in chronic inflammation, including rheumatoid arthritis (RA) synovial tissue (ST), often contain high endothelial venules and follicular dendritic cells (FDCs). Endothelial cell (EC)-specific lymphotoxin β (LTβ) receptor signaling is critical for the formation of lymph nodes and high endothelial venules. FDCs arise from perivascular platelet-derived growth factor receptor β(+) precursor cells (preFDCs) that require specific group 3 innate lymphoid cells (ILC3s) and LTβ for their expansion. Previously, we showed that RA ST contains ECs that express NF-κB-inducing kinase (NIK), which is pivotal in LTβ-induced noncanonical NF-κB signaling. We studied the relation between NIK(+) ECs, (pre)FDCs, and ILC3s with respect to TLSs in RA ST. TLS(+) tissues exhibited a significantly increased expression of genes involved in noncanonical NF-κB signaling, including NIK, and immunohistochemical analysis revealed that NIK was almost exclusively expressed by ECs. ILC3s were present in human RA ST in very low numbers, but not differentially in TLS(+) tissues. In contrast, TLS(+) tissues contained significantly more NIK(+) ECs and perivascular platelet-derived growth factor receptor β(+) preFDCs, which correlated significantly with the quantity of FDCs. We established a strong link between NIK(+) ECs, (pre)FDCs, and the presence of TLSs, indicating that NIK(+) ECs may not only be important orchestrators of lymph node development but also contribute to the formation of TLSs in chronic inflammatio