Karin M. Danzer

Different Species of α-Synuclein Oligomers Induce Calcium Influx and Seeding

Aggregation of α-synuclein (α-syn) has been linked to the pathogenesis of Parkinsons disease (PD) and other neurodegenerative diseases. Increasing evidence suggests that prefibrillar oligomers and protofibrils, rather than mature fibrils of α-syn, are the pathogenic species in PD. Despite extensive effort on studying oligomerization of α-syn, no studies have compared different oligomer species directly on a single-particle level and investigated their biological effects on cells. In this study, we applied a novel highly sensitive single molecule detection system that allowed a direct comparison of different oligomer types. Furthermore, we studied biological effects of different oligomer types on cells. For this purpose, we developed new oligomerization protocols, that enabled the use of these different oligomers in cell culture. We found that all of our three aggregation protocols resulted in heterogeneous populations of oligomers. Some types of oligomers induced cell death via disruption of cellular ion homeostasis by a presumably pore-forming mechanism. Other oligomer types could directly enter the cell resulting in increased α-syn aggregation. Based on our results, we propose that under various physiological conditions, heterogeneous populations of oligomeric forms will coexist in an equilibrium. These different oligomer types lead directly or indirectly to cell damage. Our data indicate that inhibition of early α-syn aggregation events would consequently prevent all α-syn oligomer related toxicities. This has important implications for the development of disease-modifying drugs for the treatment of PD and other synucleinopathies.

Exosomal cell-to-cell transmission of alpha synuclein oligomers

BackgroundAggregation 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.ResultsUsing 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.ConclusionsOur 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.

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

Heat-shock protein 70 modulates toxic extracellular α-synuclein oligomers and rescues trans-synaptic toxicity

The paradoxical appearance of aggregated α‐synuclein (αsyn) in naive transplanted embryonic stem cells in Parkinsons disease (PD) brains has recently been reported, highlighting the possibility of neuron to neuron transmission of αsyn in PD. Here, we demonstrate in a cellular model the presence of αsyn oligomers in the extracellular space, their uptake by neurons, retrograde axonal transport to cell soma, and detrimental effects on neighboring cells. Moreover, we demonstrate that Hsp70 chaperones αsyn in the extracellular space and reduces extracellular αsyn oligomer formation and related toxicity. These novel findings provide evidence that extracellular αsyn oligomers may represent a crucial player in the propagation of pathology in PD, with their modulation by Hsp70 representing a potential new target for therapeutic interventions.—Danzer, K. M., Ruf, W. P., Putcha, P., Joyner, D., Hashimoto, T., Glabe, C., Hyman, B. T., McLean, P. J. Heat‐shock protein 70 modulates toxic extracellular α‐synuclein oligomers and rescues trans‐synaptic toxicity. FASEBJ. 25, 326–336 (2011). www.fasebj.org

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 Parkinsons 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.

TDP-43 is intercellularly transmitted across axon terminals

A protein complementation assay quantifying TDP-43 oligomerization in living neurons shows microvesicular and bidirectional synaptic transmission of TDP-43 and TDP-43 seeding activity in human ALS postmortem brain tissue.

Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson’s disease and dementia with Lewy bodies

Stuendl et al. show that CSF exosomes of patients with Parkinson’s disease or dementia with Lewy bodies contain α-synuclein and induce α-synuclein aggregation in a reporter cell line. Thus, exosomes may support inter-neuronal transmission of α-synuclein pathology. CSF exosomal α-synuclein may serve as a biomarker in α-synuclein-related neurodegeneration.

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

Inflammatory dysregulation of blood monocytes in Parkinson’s disease patients

Despite extensive effort on studying inflammatory processes in the CNS of Parkinson’s disease (PD) patients, implications of peripheral monocytes are still poorly understood. Here, we set out to obtain a comprehensive picture of circulating myeloid cells in PD patients. We applied a human primary monocyte culture system and flow cytometry-based techniques to determine the state of monocytes from PD patients during disease. We found that the classical monocytes are enriched in the blood of PD patients along with an increase in the monocyte-recruiting chemoattractant protein CCL2. Moreover, we found that monocytes from PD patients display a pathological hyperactivity in response to LPS stimulation that correlates with disease severity. Inflammatory pre-conditioning was also reflected on the transcriptome in PD monocytes using next-generation sequencing. Further, we identified the CD95/CD95L as a key regulator for the PD-associated alteration of circulating monocytes. Pharmacological neutralization of CD95L reverses the dysregulation of monocytic subpopulations in favor of non-classical monocytes. Our results suggest that PD monocytes are in an inflammatory predisposition responding with hyperactivation to a “second hit”. These results provide the first direct evidence that circulating human peripheral blood monocytes are altered in terms of their function and composition in PD patients. This study provides insights into monocyte biology in PD and establishes a basis for future studies on peripheral inflammation.

PGC-1α is a male-specific disease modifier of human and experimental amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 3-5 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1α, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1α leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1α-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1α as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1α in this neurodegenerative disorder.