Aggregation of ␣-synuclein (␣-syn) has been linked to the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases. Increasing evidence suggests that prefibrillar oligomers and protofibrils, rather than mature fibrils of ␣-syn, are the pathogenic species in PD. Despite extensive effort on studying oligomerization of ␣-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 ␣-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 ␣-syn aggregation events would consequently prevent all ␣-syn oligomer related toxicities. This has important implications for the development of disease-modifying drugs for the treatment of PD and other synucleinopathies.

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.

The mRNAs of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) exhibit a similar, though not identical, regional and cellular distribution in the rodent brain. In situ hybridization experiments have shown that BDNF, like NGF, is predominantly expressed by neurons. The neuronal localization of the mRNAs of these two neurotrophic molecules raised the question as to whether neuronal activity might be involved in the regulation of their synthesis. After we had demonstrated that depolarization with high potassium (50 mM) resulted in an increase in the levels of both BDNF and NGF mRNAs in cultures of hippocampal neurons, we investigated the effect of a large number of transmitter substances. Kainic acid, a glutamate receptor agonist, was by far the most effective in increasing BDNF and NGF mRNA levels in the neurons, but neither N‐methyl‐D‐aspartic acid (NMDA) nor inhibitors of the NMDA glutamate receptors had any effect. However, the kainic acid mediated increase was blocked by antagonists of non‐NMDA receptors. Kainic acid also elevated levels of BDNF and NGF mRNAs in rat hippocampus and cortex in vivo. These results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non‐NMDA glutamate receptors.

The neuropathology of Parkinson's disease (PD) is characterized by the presence of a-synuclein (a-syn) inclusions. In affected neurons, the inclusions appear as thread-like Lewy neurites or globular Lewy bodies. Vulnerable neurons with Lewy pathology all belong to the class of projection neurons with long unmyelinated or poorly myelinated neurites (Braak et al. 2006).The degeneration of neuromelanin-containing dopaminergic neurons in the substantia nigra is regarded as the most important hallmark of PD and to be the primary cause of the motor deficits in PD (Dauer and Przedborski 2003). However, the neuropathology is not restricted to the substantia nigra and includes various specific, extra-nigral brain regions such as the dorsal IX/X motor nucleus, the reticulate zone, subnuclei of the Raphe system, the thalamus or amygdala and the cortex (Braak et al. 1998(Braak et al. , 2003aBraak and Braak 2000). The temporal and topographical order of the histopathological lesions in sporadic PD has been described in detail (Braak et al. 2003a,b). Braak et al. suggested a gradually progressing, ascending course with little interindividual deviation in the development of the PD neuropathology. According to this hypothesis it is conceivable that after an initial event, the disease pathology progresses without remission until reaching a terminal phase (Braak et al. 2003a(Braak et al. , 2006. Recent studies have highlighted the possibility that a seeding-nucleation mechanism may exist by studying the fate of neurons grafted into the brains of patients with PD. In these studies, grafted healthy neurons gradually developed the same pathology as the host neurons in the diseased brains (Kordower et al. 2008;Li et al. 2008; Received July 18, 2009; accepted July 28, 2009 AbstractLewy bodies, a-synuclein (a-syn) immunopositive intracellular deposits, are the pathological hallmark of Parkinson's disease (PD). Interestingly, Lewybody-like structures have been identified in fetal tissue grafts about one decade after transplantation into the striatum of PD patients. One possible explanation for the accelerated deposition of a-syn in the graft is that the aggregation of a-syn from the host tissue to the graft is spread by a prion disease-like mechanism. We discuss here an in vitro model which might recapitulate some aspects of disease propagation in PD. We found here that in vitro-generated a-syn oligomers induce transmembrane seeding of a-syn aggregation in a dose-and time-dependent manner. This effect was observed in primary neuronal cultures as well as in neuronal cell lines. The seeding oligomers were characterized by a distinctive lithium dodecyl sulfate-stable oligomer pattern and could be generated in a dynamic process out of pore-forming oligomers. We propose that a-syn oligomers form as a dynamic mixture of oligomer types with different properties and that a-syn oligomers can be converted into different types depending on the brain milieu conditions. Our data indicate that extracellular a-syn oligomers can induce intracellular...

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1345-4) contains supplementary material, which is available to authorized users.