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Abstract Background It is now realized that Parkinson’s disease (PD) pathology extends beyond the substantia nigra, affecting both central and peripheral nervous systems, and exhibits a variety of non-motor symptoms often preceding motor features. Neuroinflammation induced by activated microglia and astrocytes is thought to underlie these manifestations. α-Synuclein aggregation has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration; however, there is still a lack of critical information about the structural identity of the α-synuclein conformers that activate microglia and/or astrocytes and the molecular pathways involved. Methods To investigate the role of α-synuclein conformers in the development and maintenance of neuroinflammation, we used primary quiescent microglia and astrocytes, post-mortem brain tissues from PD patients and A53T α-synuclein transgenic mice that recapitulate key features of PD-related inflammatory responses in the absence of cell death, i.e., increased levels of pro-inflammatory cytokines and complement proteins. Biochemical and -omics techniques including RNAseq and secretomic analyses, combined with 3D reconstruction of individual astrocytes and live calcium imaging, were used to uncover the molecular mechanisms underlying glial responses in the presence of α-synuclein oligomers in vivo and in vitro. Results We found that the presence of SDS-resistant hyper-phosphorylated α-synuclein oligomers, but not monomers, was correlated with sustained inflammatory responses, such as elevated levels of endogenous antibodies and cytokines and microglial activation. Similar oligomeric α-synuclein species were found in post-mortem human brain samples of PD patients but not control individuals. Detailed analysis revealed a decrease in Iba1Low/CD68Low microglia and robust alterations in astrocyte number and morphology including process retraction. Our data indicated an activation of the p38/ATF2 signaling pathway mostly in microglia and a sustained induction of the NF-κB pathway in astrocytes of A53T mice. The sustained NF-κB activity triggered the upregulation of astrocytic T-type Cav3.2 Ca2+ channels, altering the astrocytic secretome and promoting the secretion of IGFBPL1, an IGF-1 binding protein with anti-inflammatory and neuroprotective potential. Conclusions Our work supports a causative link between the neuron-produced α-synuclein oligomers and sustained neuroinflammation in vivo and maps the signaling pathways that are stimulated in microglia and astrocytes. It also highlights the recruitment of astrocytic Cav3.2 channels as a potential neuroprotective mediator against the α-synuclein-induced neuroinflammation. Graphical Abstract

α-Synuclein (α-syn) has been genetically and biochemically linked to the pathogenesis of Parkinson’s disease (PD). There is accumulating evidence that misfolded α-syn species spread between cells in a prion-like manner and seed the aggregation of endogenous protein in the recipient cells. Exosomes have been proposed to mediate the transfer of misfolded α-syn and thus facilitate disease transmission, although the pathological mechanism remains elusive. Here, we investigated the seeding capacity of exosome-associated α-syn, in vivo. Disease-associated α-syn was present in exosome fractions isolated from transgenic A53T mouse brain. However, following intrastriatal injection of such exosomes in wild-type (wt) mice, we were not able to detect any accumulation of endogenous α-syn. In addition, recombinant fibrillar α-syn, when loaded to isolated brain exosomes, induced minor pathological α-syn brain accumulation at 7 months post injection. These data suggest that exosomes neutralize the effect of toxic α-syn species and raise additional questions on their paracrine modulatory role in disease transmission.

Abstract In experimental models, both in vivo and cellular, over-expression of Parkinson’s linked mutant leucine-rich repeat kinase 2 (LRRK2) is sufficient to induce neuronal death. While several cell death associated proteins have been linked to LRRK2, either as protein interactors or as putative substrates, characterization of the neuronal death cascade remains elusive. In this study, we have mapped for the first time the domain within LRRK2 that mediates the interaction with FADD, thereby activating the molecular machinery of the extrinsic death pathway. Using homology modeling and molecular docking approaches, we have identified a critical motif within the N-terminal armadillo repeat region of LRRK2. Moreover, we show that co-expression of fragments of LRRK2 that contain the FADD binding motif, or deletion of this motif itself, blocks the interaction with FADD, and is neuroprotective. We further demonstrate that downstream of FADD, the mitochondrial proteins Bid and Bax are recruited to the death cascade and are necessary for neuronal death. Our work identifies multiple novel points within neuronal death signaling pathways that could potentially be targeted by candidate therapeutic strategies and highlight how the extrinsic pathway can be activated intracellularly in a pathogenic context.

Alpha-synuclein (α-syn) is biochemically and genetically linked to Parkinson’s disease (PD) and other synucleinopathies. It is now widely accepted that α-syn can be released in the extracellular space, even though the mechanism of its release is still unclear. In addition, pathology-related aggregated species of α-syn have been shown to propagate between neurons in synaptically connected areas of the brain thereby assisting the spreading of pathology in healthy neighboring neuronal cells. In neurons, calcium channels are key signaling elements that modulate the release of bioactive molecules (hormones, proteins, and neurotransmitters) through calcium sensing. Such calcium sensing activity is determined by the distinct biophysical and pharmacological properties and the ability of calcium channels to interact with other modulatory proteins. Although the function of extracellular α-syn is currently unknown, previous work suggested the presence of a calcium-dependent mechanism for α-syn secretion both in vitro, in neuronal cells in culture, and also in vivo, in the context of a trans-neuronal network in brain. Mechanisms regulating extracellular α-syn levels may be of particular importance as they could represent novel therapeutic targets. We discuss here how calcium channel activity may contribute to α-syn aggregation and secretion as a pathway to disease progression in synucleinopathies.

Despite the plethora of sequence variants in LRRK2, only a few clearly segregate with PD. Even within this group of pathogenic mutations, the phenotypic profile can differ widely.We examined multiple properties of LRRK2 behavior in cellular models over-expressing three sequence variants described in Greek PD patients in comparison to several known pathogenic and non-pathogenic LRRK2 mutations, to determine if specific phenotypes associated with pathogenic LRRK2 can be observed in other less-common sequence variants for which pathogenicity is unclear based on clinical and/or genetic data alone.The oligomerization, activity, phosphorylation, and interaction with FADD was assessed in HEK293T cells over-expressing LRRK2; while the induction of neuronal death was determined by quantifying apoptotic nuclei in primary neurons transiently expressing LRRK2.One LRRK2 variant, A211V, exhibited a modest increase in kinase activity, whereas only the pathogenic mutants G2019S and I2020T displayed significantly altered auto-phosphorylation. We observed an induction of detergent-insoluble high molecular weight structures upon expression of pathogenic LRRK2 mutants, but not the other LRRK2 variants. In contrast, each of the variants tested induced apoptotic death of cultured neurons similar to pathogenic LRRK2 in a FADD-dependent manner.Overall, despite differences in some properties of LRRK2 function such as kinase activity and its oligomerization, each of the LRRK2 variants examined induced neuronal death to a similar extent. Furthermore, our findings further strengthen the notion of a convergence on the extrinsic cell death pathway common to mutations in LRRK2 that are capable of inducing neuronal death.