**4. Conclusion**

182 Etiology and Pathophysiology of Parkinson's Disease

finally, to be degraded by lysosomes (H. J. Lee et al., 2008). However, these results were obtained by using very high concentrations of recombinant α-synuclein and cationic

Importantly, recent data using cell-secreted α-synuclein have verified its impact on neuronal survival. Application of conditioned medium containing cell-secreted αsynuclein to neuronal cells induced cell death to the recipient cells (Emmanouilidou, Melachroinou et al., 2010). This toxic effect was concentration-dependent and was conferred synergistically by both oligomeric and monomeric α-synuclein species present in the conditioned medium. In this study, however, there was evidence of very low, if any, α-synuclein uptake by neuronal cells (Emmanouilidou, Melachroinou et al., 2010). Similarly, apoptotic death of neurons, both *in vitro* and *in vivo*, was observed upon their exposure to cell-derived extracellular α-synuclein (Desplats et al., 2009). Secreted αsynuclein, that was readily endocytosed by neurons, was transmitted from one cell to another thereby supporting the idea of a mechanism of pathological propagation in PD (Desplats et al., 2009). Cell-to-cell transfer of α-synuclein was also demonstrated using coculture systems (Hansen et al., 2010). In fact, this transfer did not require cell contact and was independent of the aggregation state of the protein. Fluorescently-labeled recombinant α-synuclein was uptaken by neuronal cells *in vitro* and *in vivo* via an endocytic mechanism. Altogether, these data demonstrated that endocytosed extracellular α-synuclein can be internalized by recipient cells, interact with the pool of intracellular α-

An alternative mechanism of neurodegeneration induced by extracellular α-synuclein may involve the initiation of neuroinflammatory responses. Microglia are resident immune cells that are sensitive to even minor disturbances in the homeostasis of the central nervous system (Soulet & Rivest, 2008). Activation of microglia results in a change in cell morphology (from a ramified to amoeboid shape) accompanied by alterations in surface receptor expression, production of reactive oxygen species (ROS) and release of chemokines and cytokines (Kim & Joh, 2006; Soulet & Rivest, 2008). There is increasing evidence suggesting that extracellularly added recombinant α-synuclein can trigger microglia activation which induces the production of various cytokines, such as IL1β and IL6, and inflammation-related enzymes (Su et al., 2008; Zhang et al., 2005). In fact, microglia activation has been shown to be one of the mechanisms by which α-synuclein induces dopaminergic neurodegeneration, rather than being an epiphenomenon following cell death (Zhang et al., 2007). Further dissection of the pathway of microglia activation, suggested that α-synuclein potentially binds to Mac-1 receptors which subsequently activate PHOX, a ROS-generating enzyme, to produce O2• ultimately leading to neurotoxicity. Importantly, microglia activation did not require internalization/phagocytosis of α-synuclein by microglial cells (Zhang et al., 2007).To this end, microglial prostaglandin E2 receptor subtype 2 (EP2) plays a critical role in αsynuclein-induced neurotoxicity partly by decreasing PHOX activation (Jin et al., 2007). Cell-produced α-synuclein also resulted in the activation of primary microglia, leading to the induction of inflammatory signaling pathways (E. J. Lee et al., 2010). It was suggested that α-synuclein-induced microglia activation involves the secretion of MMPs which in turn activate PAR-1 receptor (E. J. Lee et al.). Alternatively, recent data indicate that cellreleased α-synuclein can also be internalized by astrocytes thereby producing

liposomes to assist the uptake.

synuclein and seed aggregation (Hansen et al., 2010).

inflammatory responses both *in vitro* and *in vivo* (E. J. Lee et al., 2010).

α-Synuclein is genetically linked to PD. Maintenance of intracellular steady-state concentration of α-synuclein is considered to be a key challenge for neuronal homeostasis and total levels of the protein have been directly linked with PD pathogenesis. Importantly, Genome-Wide association Studies (GWAS) have provided a strong genetic link between αsynuclein and sporadic PD, and clearly point to α-synuclein as being one of the very few genetic loci consistently associated with disease progression. The physiological and aberrant functions of α-synuclein are still under investigation. However, cytoplasmic soluble oligomers/protofibrils of the protein appear to be one of the primary "suspects" in the pathogenesis of PD. Therefore, prevention of α-synuclein aggregation and intervention in the mechanisms of abnormal protein turnover appears to be a highly promising therapeutic target for the treatment of PD as well as other synucleinopathies.

From a therapeutic standpoint, it follows that enhancement of α-synuclein clearance via proteasomal or lysosomal degradation may represent a valid therapeutic intervention for PD. New evidence, suggests that α-synuclein is also physiologically secreted, and as such, it can exert as yet unknown paracrine effects in the brain. Still, the presence and exact levels of α-synuclein in the interstitial fluid in the brain remain to be clarified. Recent clinical observations have suggested that secreted α-synuclein may aggravate PD pathology via a mechanism that underlies cell-to-cell propagation of the protein. It is possible that a dynamic equilibrium between intracellular and extracellular α-synuclein exists, ensuring normal function of neuronal cells. In this respect, dysfunctions in the mechanism(s) regulating extracellular α-synuclein levels, such as mechanisms of secretion or extracellular clearance, may affect neuronal survival. Increases in extracellular α-synuclein may trigger the formation of toxic oligomers in neighbouring neurons and in the extracellular space, and result in inflammatory glia activation, utterly leading to a vicious cycle of neurodegeneration. Along these lines, compounds which block other signalling pathways switched on as a consequence of microglial activation which may ultimately lead to neuronal death- might also represent new targets for therapeutic intervention. Under this scope, manipulation of regulatory mechanisms that alleviate the extracellular α-synuclein "burden" represents a potential target for the development of novel treatment strategies for PD. It is obvious that α-synuclein can affect neuronal cell homeostasis in numerous ways and at multiple levels. The intrinsic complexity of the neuronal interface may suggest that its actions be considered within the context of non cell-autonomous models and thus be interpreted by taking into account that the nature of communication between brain cells is indeed very dynamic.
