**3. Effects of extracellular** α**-synuclein on cellular homeostasis**

### **3.1** α**-synuclein is detected in biological fluids**

Since α-synuclein lacks a signal peptide targeting the protein for ER-mediated exocytosis, it was considered to be primarily localized in the cytoplasm where it would exert its pathogenic effects. However, a number of studies suggest that α-synuclein can be secreted in the medium of cultured cells and is detectable in human biological fluids such as CSF and plasma of PD patients and controls. The first studies to demonstrate detection of α-synuclein in the CSF and plasma utilized biochemical techniques such as immunoprecipitation and western blotting in a small number of human samples (Borghi et al., 2000; El-Agnaf et al., 2003). However, these initial studies failed to show a significant difference in the levels of αsynuclein between PD and healthy subjects.

**19S binding**

**I II III**

Since α-synuclein lacks a signal peptide targeting the protein for ER-mediated exocytosis, it was considered to be primarily localized in the cytoplasm where it would exert its pathogenic effects. However, a number of studies suggest that α-synuclein can be secreted in the medium of cultured cells and is detectable in human biological fluids such as CSF and plasma of PD patients and controls. The first studies to demonstrate detection of α-synuclein in the CSF and plasma utilized biochemical techniques such as immunoprecipitation and western blotting in a small number of human samples (Borghi et al., 2000; El-Agnaf et al., 2003). However, these initial studies failed to show a significant difference in the levels of α-

Fig. 2. Schematic illustration of the possible interaction between α-synuclein and the 26S proteasome. α-synuclein can gain access into the interior of the 20S champer and bind to one or more of the catalytic β-subunits. Such changes would interfere with the active site(s) thus decreasing the overall proteolytic activity of the proteasome (I). Alternatively, oligomeric αsynuclein can directly bind to a subunit of the 19S complex thereby inhibiting substrate recognition or gate opening (II). Finally, α-synuclein oligomers can transiently interact with the 19S particle and its function possibly by preventing 19S subunits from obtaining the appropriate conformation. This "clogging" of the 19S cap would result in the cytoplasmic

accumulation of other proteins due to their deficient degradation (III).

**3.1** α**-synuclein is detected in biological fluids** 

synuclein between PD and healthy subjects.

**3. Effects of extracellular** α**-synuclein on cellular homeostasis** 

**20S**

**19S transient association**

α**-synuclein oligomers**

**unfolding**

**19S**

**translocation**

**20S binding**

In an attempt to assess the applicability of α-synuclein concentration in biological fluids as a biomarker for PD, α-synuclein was measured by specific ELISAs that provide higher sensitivity and accuracy. Some of these studies (Mollenhauer et al., 2008; Tokuda et al., 2006), but not all (Ohrfelt et al., 2009), reported significant differences in the levels of αsynuclein in PD and control samples. Since there is substantial evidence indicating that αsynuclein aggregation is central in PD pathogenesis, some other studies focus on the quantification of α-synuclein oligomers in CSF (Tokuda et al., 2010) or plasma (El-Agnaf et al., 2006) using oligomer-specific ELISAs. Overall, there is great variability in the amount of α-synuclein quantified in either blood plasma or CSF. Two basic reasons could account for this discrepancy. First, the ELISA system employed for the measurement of α-synuclein varies between groups in terms of both the antibodies and the detection method used. This results in differences in the specificity and the sensitivity of the measurement. Second, each group does not follow similar protocols for sample collection and processing. Protein integrity and erythrocyte contamination are important parameters related to sample acquisition and processing and should be carefully monitored to assure valid assessment of α-synuclein in biological fluids. While future work is required to establish a correlation between disease and α-synuclein levels in biological fluids, α-synuclein remains an appealing protein to be used as a diagnostic marker for PD.

#### **3.2 Mechanism of** α**-synuclein release**

Numerous studies employing a variety of cell systems reveal a dynamic network of molecular communication between cells, involving secretion. Deciphering the components of this network as well as their biological significance represents a major challenge in the field of neurodegeneration in particular.

In this respect, α-synuclein has been shown to be released from neuronal cells in culture independently of the expression method used; stable overexpression (El-Agnaf et al., 2003), inducible overexpression (Emmanouilidou, Melachroinou et al., 2010), transient transfection (Sung et al., 2005) or viral-mediated expression (H. J. Lee, Patel, & Lee, 2005). The presence of α-synuclein in the conditioned media (CM) of the α-synuclein-expressing cells reflects physiologic secretion of the protein and not an artifact of membrane leakage, since other abundant cytoplasmic proteins are not detected in the CM. The secretion of α-synuclein has been reported to be insensitive to brefeldin A (H. J. Lee et al., 2005), suggesting that it is secreted via an ER/Golgi-independent pathway. In accordance with a vesicular mechanism of secretion, a portion of intracellular α-synuclein has been found in the lumen of vesicles from rat brain homogenates, rat embryonic cortical neurons and human neuroblastoma cells (H. J. Lee et al., 2005). Electron microscopy and density gradient ultracentrifugation suggested that the vesicles containing α-synuclein have morphologies and sedimentation properties similar to the dense core vesicles (H. J. Lee et al., 2005), but their exact identities remain unknown.

In a recent study, treatment of MES cells in culture with aggregated recombinant αsynuclein results in the internalization of the protein which is subsequently released in the extracellular space by rab11a/HSP90-mediated exocytosis (Liu et al., 2009). The mechanism of exocytosis was found to be temperature-sensitive and time-dependent. Part of this internalized protein is also degraded through the lysosomal-endosomal pathway (Liu et al., 2009). Indeed, we recently demonstrated that a non-classical secretory pathway is involved in the physiological and constitutive release of α-synuclein in the extracellular space

Effects of Alpha-Synuclein on Cellular Homeostasis 179

signifies selectivity of protein sequestration (Vella, Sharples, Nisbet, Cappai, & Hill, 2008). Interestingly, this bi-layer also carries transmembrane cell adhesion molecules such as integrins, which enable the dynamic communication of the cytoskeleton with the extracellular matrix (ECM) and neighboring cells. The origin of exosomes led to the suggestion that this mechanism was an alternative to autophagic degradation, another means of "discarding" unwanted cytosolic material. Recently, it was found that under certain conditions, exosomes can be biologically active entities, important for intercellular communication (Valadi et al., 2007) and key players in significant biological processes. They are secreted by most cells that have been examined so far including primary neurons (Lachenal et al., 2010). Furthermore, exosomal release by neurons was shown to be dependent on synaptic activity. It is suggested that exosomes could be a mechanism of releasing proteins in the extracellular space in order to be proteolytically processed. Alternatively, exosomes can mediate cell-to-cell communication since they can attach and fuse with membranes of neighbouring target cells transferring exosomal molecules from one cell to another (Thery et al., 2002; Vella et al., 2008). The exosomal pathway thus seems to represent a well-designed mechanism for local and systemic

Several groups have reported that exosomes contain pathological proteins. Biochemical studies from L. Rajendran et al. in 2006 reported that Aβ peptides were indeed found on vesicles positive for specific markers of exosomal identity (Rajendran et al., 2006). This suggested that toxic species of processed Amyloid beta Precursor Protein (APP) are also excreted via exosomes. Most importantly, neuritic plaques are co-localized with exosomal markers, indicating that exosomes are able to act at a distance from their source of generation like amyloidogenic fragments of the APP (Rajendran et al., 2006). Similarly, Fevrier and Raposo demonstrated association of prion protein with exosomes (Fevrier & Raposo, 2004). In this sense, exosomes are the central component of a theory that is starting to gain scientific traction over the past few years. The "Trojan horse" hypothesis is an appealing hypothesis according to which, toxic protein contents of a cell are packed into exosomes, shipped extracellularly and are subsequently received by neighboring cells in the context of cell-to-cell communication (Ghidoni, Benussi, & Binetti, 2008). Upon membrane fusion, exosomal cargo is released and causes spread of disease. Up-regulation of exosome secretion is correlated with conditions that promote protein misfolding and impair proteolysis (Alvarez-Erviti et al.; Eldh et al.; Jang et al.), hence, increase cytosolic cargo of a particular protein. In our study, α-synuclein was shown to be associated with both the exosomal membrane and lumen. Importantly, not only monomeric α-synuclein but also oligomeric forms of the protein were found in our exosomal preparations (Emmanouilidou, Melachroinou et al., 2010), further suggesting that exosomes can indeed carry "potentially toxic" cargo. The finding that α-synuclein can be partly externalized via the exosomal pathway provides a common mechanism for the delivery of a potentially cytotoxic protein

Undoubtedly, deciphering networks of intercellular communication is a fascinating field of research. Understanding the physiological mechanisms of exchanging information between cells will allow the identification of new, effective therapeutic targets for late-onset neurodegenerative diseases, including Parkinson's disease. The dynamic nature of neuronto-neuron interactions leads us to the thought that more enlightening answers are to come from the field of synaptic plasticity and function. So far, data interpretation in most studies focuses on cell-autonomous effects and networks. Perhaps, data interpretation should be

inter-neuronal transfer of information (Smalheiser, 2007).

in the extracellular space (Figure 3).

(Emmanouilidou, Melachroinou et al., 2010). In this study, α-synuclein was exported in a calcium-dependent manner in association with externalized membrane vesicles that involved in the endocytic pathway (Figure 3).

Fig. 3. α-Synuclein transportation through the endocytic pathway. Along with membrane proteins, secreted α-synuclein can enter the cell via endocytosis of clathrin-coated vesicles which fuse with early endosomes. In early endosomes, protein material is either recycled back to the plasma membrane or sorted to MVBs. Cytoplasmic α-synuclein can also enter MVBs at this point via inward budding of the limiting membrane of these vesicles. For protein degradation, MVBs fuse with lysosomes. Alternatively, MVBs can fuse with the plasma membrane releasing their content in the extracellular space as exosomes.

In the first step of the endocytic pathway (Figure 3) internalized proteins via clathrin-coated vesicles are delivered to early endosomes. Proteins are then either recycled back to the plasma membrane or accumulate in multivesicular endosomes, commonly called multivesicular bodies (MVBs). Proteins destined for degradation are sorted into small (40- 100 nm in diameter) intraluminal vesicles (ILVs) that are generated by inward budding from the limiting membrane of MVBs (Fevrier & Raposo, 2004; Keller, Sanderson, Stoeck, & Altevogt, 2006). Degradation of the vesicle-associated proteins and lipids is achieved upon fusion of the MVBs with lysosomes. This process allows the cell to remove certain transmembrane proteins and excessive membranes. Alternatively, MVBs can fuse with the plasma membrane releasing ILVs in the extracellular environment as exosomes (Fevrier & Raposo, 2004; Keller et al., 2006).

Exosomal protein content includes cytosolic proteins, heat shock proteins, tetraspanins and transmembrane proteins; proteins originating from mitochondria, ER or nucleus are excluded (Thery, Zitvogel, & Amigorena, 2002). Exosomes share common characteristics, most important of which is that they are delimited in a cholesterol-rich lipid bi-layer containing cytosolic compounds. Most secreted exosomes contain lipid rafts, a characteristic which

(Emmanouilidou, Melachroinou et al., 2010). In this study, α-synuclein was exported in a calcium-dependent manner in association with externalized membrane vesicles that

Fig. 3. α-Synuclein transportation through the endocytic pathway. Along with membrane proteins, secreted α-synuclein can enter the cell via endocytosis of clathrin-coated vesicles which fuse with early endosomes. In early endosomes, protein material is either recycled back to the plasma membrane or sorted to MVBs. Cytoplasmic α-synuclein can also enter MVBs at this point via inward budding of the limiting membrane of these vesicles. For protein degradation, MVBs fuse with lysosomes. Alternatively, MVBs can fuse with the plasma membrane releasing their content in the extracellular space as exosomes.

In the first step of the endocytic pathway (Figure 3) internalized proteins via clathrin-coated vesicles are delivered to early endosomes. Proteins are then either recycled back to the plasma membrane or accumulate in multivesicular endosomes, commonly called multivesicular bodies (MVBs). Proteins destined for degradation are sorted into small (40- 100 nm in diameter) intraluminal vesicles (ILVs) that are generated by inward budding from the limiting membrane of MVBs (Fevrier & Raposo, 2004; Keller, Sanderson, Stoeck, & Altevogt, 2006). Degradation of the vesicle-associated proteins and lipids is achieved upon fusion of the MVBs with lysosomes. This process allows the cell to remove certain transmembrane proteins and excessive membranes. Alternatively, MVBs can fuse with the plasma membrane releasing ILVs in the extracellular environment as exosomes (Fevrier &

Exosomal protein content includes cytosolic proteins, heat shock proteins, tetraspanins and transmembrane proteins; proteins originating from mitochondria, ER or nucleus are excluded (Thery, Zitvogel, & Amigorena, 2002). Exosomes share common characteristics, most important of which is that they are delimited in a cholesterol-rich lipid bi-layer containing cytosolic compounds. Most secreted exosomes contain lipid rafts, a characteristic which

involved in the endocytic pathway (Figure 3).

Raposo, 2004; Keller et al., 2006).

signifies selectivity of protein sequestration (Vella, Sharples, Nisbet, Cappai, & Hill, 2008). Interestingly, this bi-layer also carries transmembrane cell adhesion molecules such as integrins, which enable the dynamic communication of the cytoskeleton with the extracellular matrix (ECM) and neighboring cells. The origin of exosomes led to the suggestion that this mechanism was an alternative to autophagic degradation, another means of "discarding" unwanted cytosolic material. Recently, it was found that under certain conditions, exosomes can be biologically active entities, important for intercellular communication (Valadi et al., 2007) and key players in significant biological processes. They are secreted by most cells that have been examined so far including primary neurons (Lachenal et al., 2010). Furthermore, exosomal release by neurons was shown to be dependent on synaptic activity. It is suggested that exosomes could be a mechanism of releasing proteins in the extracellular space in order to be proteolytically processed. Alternatively, exosomes can mediate cell-to-cell communication since they can attach and fuse with membranes of neighbouring target cells transferring exosomal molecules from one cell to another (Thery et al., 2002; Vella et al., 2008). The exosomal pathway thus seems to represent a well-designed mechanism for local and systemic inter-neuronal transfer of information (Smalheiser, 2007).

Several groups have reported that exosomes contain pathological proteins. Biochemical studies from L. Rajendran et al. in 2006 reported that Aβ peptides were indeed found on vesicles positive for specific markers of exosomal identity (Rajendran et al., 2006). This suggested that toxic species of processed Amyloid beta Precursor Protein (APP) are also excreted via exosomes. Most importantly, neuritic plaques are co-localized with exosomal markers, indicating that exosomes are able to act at a distance from their source of generation like amyloidogenic fragments of the APP (Rajendran et al., 2006). Similarly, Fevrier and Raposo demonstrated association of prion protein with exosomes (Fevrier & Raposo, 2004). In this sense, exosomes are the central component of a theory that is starting to gain scientific traction over the past few years. The "Trojan horse" hypothesis is an appealing hypothesis according to which, toxic protein contents of a cell are packed into exosomes, shipped extracellularly and are subsequently received by neighboring cells in the context of cell-to-cell communication (Ghidoni, Benussi, & Binetti, 2008). Upon membrane fusion, exosomal cargo is released and causes spread of disease. Up-regulation of exosome secretion is correlated with conditions that promote protein misfolding and impair proteolysis (Alvarez-Erviti et al.; Eldh et al.; Jang et al.), hence, increase cytosolic cargo of a particular protein. In our study, α-synuclein was shown to be associated with both the exosomal membrane and lumen. Importantly, not only monomeric α-synuclein but also oligomeric forms of the protein were found in our exosomal preparations (Emmanouilidou, Melachroinou et al., 2010), further suggesting that exosomes can indeed carry "potentially toxic" cargo. The finding that α-synuclein can be partly externalized via the exosomal pathway provides a common mechanism for the delivery of a potentially cytotoxic protein in the extracellular space (Figure 3).

Undoubtedly, deciphering networks of intercellular communication is a fascinating field of research. Understanding the physiological mechanisms of exchanging information between cells will allow the identification of new, effective therapeutic targets for late-onset neurodegenerative diseases, including Parkinson's disease. The dynamic nature of neuronto-neuron interactions leads us to the thought that more enlightening answers are to come from the field of synaptic plasticity and function. So far, data interpretation in most studies focuses on cell-autonomous effects and networks. Perhaps, data interpretation should be

Effects of Alpha-Synuclein on Cellular Homeostasis 181

systems. Impairement of lysosomal and proteasomal protein degradation increases the burden of uncleared, unwanted proteins thus promoting their further accumulation and the development of a self-propagating cycle that eventually leads to cell death. Lysosomal function has been reported to decrease in PD patients (Alvarez-Erviti et al.; Chu, Dodiya, Aebischer, Olanow, & Kordower, 2009) and α-synuclein has been shown to be degraded by the lysosome specific mechanism of chaperone mediated autophagy (Cuervo, Stefanis,

Interestingly, Alvarez-Erviti et al. (2010), recently demonstrated that lysosomal inhibition in cells dramatically increased the intracellular and secreted pools of α-synuclein (Alvarez-Erviti et al.). The group further demonstrated a neuron-to-neuron exchange of cytosolic content via exosomes. It could be that under conditions which promote the intracellular accumulation of misfolded proteins, such as lysosomal and proteasomal dysfunction, the homeostatic mechanisms favor the secretion of aggregated forms of α-synuclein. Although the evidence for extracellular α-synuclein internalization in Emmanouilidou (2010) and Alvarez-Erviti (2010) studies are slightly debatable, there are strong indications at both that exosomes are an important mediator of intercellular communication. Exosome exchange between neurons might also represent a way for propagating pathological alterations throughout the brain during neurodegenerative diseases (Aguzzi & Rajendran, 2009;

A demonstration that exosomes allow exchange of proteinaceaous or genetic material within the nervous system would provide an an explanation of how pathologies like Alzheimer's Creuzfeld Jacob or Parkinson's diseases, which begin in discrete regions spread overtime to connected regions of the central nervous system. This idea proposes that drugs directed toward reducing the formation and/or facilitating the clearance of misfolded α-synuclein, in order to arrest or reverse the self-propagation process, might represent novel therapeutic interventions for the treatment of PD. In addition, understanding how the neuropathology spreads throughout the nervous system in Parkinson's disease, will open up avenues for

There are several studies addressing the role of extracellular α-synuclein especially in the context of PD pathology. The first indications that high levels of extracellular α-synuclein can impact cell viability came from studies using the recombinant protein. Exogenous addition of recombinant α-synuclein to the cultured medium of neuronal cells significantly decreased the viability of the recipient cells. Cell death was linearly correlated with the concentration of exogenous α-synuclein and was amplified when the applied protein also contained soluble oligomers (Albani et al., 2004; Du et al., 2003; Sung et al., 2001; Zhang et al., 2005). Application of recombinant monomeric or aggregated α-synuclein also revealed that this protein can be readily be uptaken by neuronal cells or even neural stem cells in culture (Ahn, Kim, Kang, Ryu, & Kim, 2006; Desplats et al., 2009; H. J. Lee et al., 2008; Luk et al., 2009; Sung et al., 2001). It has been suggested that the mechanism for α-synuclein internalization involves receptor-mediated endocytosis of the protein (Desplats et al., 2009; H. J. Lee et al., 2008; Sung et al., 2001). It has been proposed that this mechanism specifically mediates the uptake of oligomeric and fibrillar α-synuclein whereas monomeric α-synuclein enters cells via simple diffusion across the plasma membrane. Following internalization, extracellular α-synuclein was shown to move through the endosomal compartment and

Fredenburg, Lansbury, & Sulzer, 2004; Xilouri et al., 2008).

**3.4 Effects of extracellular** α**-synuclein on cellular homeostasis** 

Smalheiser, 2007).

new treatments.

realized under the scope of a three-dimensional neuronal interface in order to uncover the moving forces underlying cell content alterations and communication at a systemic level. The exact role and contribution of exosomes in this dynamic interplay remains to be elucidated.

#### **3.3 Pathologic neuronal interplay mediated by** α**-synuclein?**

Recent studies by Desplats et al. demonstrated that neurons overexpressing α-synuclein can transmit the protein to neural precursor cells in tissue culture and in transgenic animals (Desplats et al., 2009). Interestingly, the precursors were shown to readily uptake and propagate α-synuclein oligomers leading to cellular dysfunction as well as to inflammatory responses. Therapeutic strategies directed at reducing the formation and propagation of αsynuclein oligomers might be critical in developing new treatments for PD and DLB. Among them, considerable effort has been devoted in the last few years to promoting the clearance. This can be achieved by increasing lysosomal activity (autophagy) or degradation with immunotherapy or by pharmacologically blocking α-synuclein aggregation with small organic molecules.

Host-to-graft propagation of α-synuclein pathology has recently been demonstrated with the discovery that fetal dopaminergic neurons (derived from multiple, genetically unrelated donors) that had been implanted into PD patients 11–14 years earlier developed Lewy body pathology immunopositive for α-synuclein and thioflavin-S (Kordower, Chu, Hauser, Freeman, & Olanow, 2008; J. Y. Li et al., 2008). It is possible that these inclusions were formed as a result of the "disease environment" of the PD brain. One plausible explanation is that oligomeric α-synuclein was transmitted from the already affected host neurons to healthy implanted fetal neurons, and induced endogenous α-synuclein to misfold. Such an infective process mechanism is supported by the Desplats et al., data and could be an explanation of the step-wise progression of the disease pathology and the involvement of specific neural pathways as suggested by the Braak staging of PD progression (Braak, Del Tredici et al., 2003). Importantly, Patric Brundin and his group recently demonstrated that this process may be indeed involved in the spread of aggregated synuclein in a manner similar to that suggested for prion diseases (J. Y. Li et al., 2008). The group showed *in vivo* and *in vitro* that α-synuclein not only can transfer from one cell to another, but also that the transferred protein can seed aggregation of α-synuclein in recipient cells. Alternatively, the source of "seeding" might be microparticles, like exosomes containing α-synuclein, which following uptake by healthy "acceptors" accelerate aggregation of endogenous α-synuclein.

Collectively, recent data provide good evidence to speculate that α-synuclein exhibits prionlike behaviour. For example, oligomers from both misfolded prion and α-synuclein can "instruct" the misfolding of the normal proteins (Ferreon, Gambin, Lemke, & Deniz, 2009). Therefore it is possible, that α-synuclein is a prion itself that in a misfolded oligomeric conformation can be transmitted to neighbouring healthy neurons, thus extending the disease process. However, the cause of such an infectious spread has to be more multifactorial. The parkinsonian milieu that causes α-synuclein accumulation and extension of pathology is not yet known and could be the result, of a combination of factors. For example, aging, oxidative stress, and inflammation, may contribute to altered metabolism of α-synuclein, resulting in the pathogenesis of sporadic PD. Furthermore, continuous accumulation of misfolded proteins ,which is a common pathological phenomenon in various neurodegenerative disorders, compromises the ability of the cell's proteolytic

realized under the scope of a three-dimensional neuronal interface in order to uncover the moving forces underlying cell content alterations and communication at a systemic level. The exact role and contribution of exosomes in this dynamic interplay remains to be

Recent studies by Desplats et al. demonstrated that neurons overexpressing α-synuclein can transmit the protein to neural precursor cells in tissue culture and in transgenic animals (Desplats et al., 2009). Interestingly, the precursors were shown to readily uptake and propagate α-synuclein oligomers leading to cellular dysfunction as well as to inflammatory responses. Therapeutic strategies directed at reducing the formation and propagation of αsynuclein oligomers might be critical in developing new treatments for PD and DLB. Among them, considerable effort has been devoted in the last few years to promoting the clearance. This can be achieved by increasing lysosomal activity (autophagy) or degradation with immunotherapy or by pharmacologically blocking α-synuclein aggregation with small

Host-to-graft propagation of α-synuclein pathology has recently been demonstrated with the discovery that fetal dopaminergic neurons (derived from multiple, genetically unrelated donors) that had been implanted into PD patients 11–14 years earlier developed Lewy body pathology immunopositive for α-synuclein and thioflavin-S (Kordower, Chu, Hauser, Freeman, & Olanow, 2008; J. Y. Li et al., 2008). It is possible that these inclusions were formed as a result of the "disease environment" of the PD brain. One plausible explanation is that oligomeric α-synuclein was transmitted from the already affected host neurons to healthy implanted fetal neurons, and induced endogenous α-synuclein to misfold. Such an infective process mechanism is supported by the Desplats et al., data and could be an explanation of the step-wise progression of the disease pathology and the involvement of specific neural pathways as suggested by the Braak staging of PD progression (Braak, Del Tredici et al., 2003). Importantly, Patric Brundin and his group recently demonstrated that this process may be indeed involved in the spread of aggregated synuclein in a manner similar to that suggested for prion diseases (J. Y. Li et al., 2008). The group showed *in vivo* and *in vitro* that α-synuclein not only can transfer from one cell to another, but also that the transferred protein can seed aggregation of α-synuclein in recipient cells. Alternatively, the source of "seeding" might be microparticles, like exosomes containing α-synuclein, which following uptake by healthy "acceptors" accelerate aggregation of endogenous α-synuclein. Collectively, recent data provide good evidence to speculate that α-synuclein exhibits prionlike behaviour. For example, oligomers from both misfolded prion and α-synuclein can "instruct" the misfolding of the normal proteins (Ferreon, Gambin, Lemke, & Deniz, 2009). Therefore it is possible, that α-synuclein is a prion itself that in a misfolded oligomeric conformation can be transmitted to neighbouring healthy neurons, thus extending the disease process. However, the cause of such an infectious spread has to be more multifactorial. The parkinsonian milieu that causes α-synuclein accumulation and extension of pathology is not yet known and could be the result, of a combination of factors. For example, aging, oxidative stress, and inflammation, may contribute to altered metabolism of α-synuclein, resulting in the pathogenesis of sporadic PD. Furthermore, continuous accumulation of misfolded proteins ,which is a common pathological phenomenon in various neurodegenerative disorders, compromises the ability of the cell's proteolytic

**3.3 Pathologic neuronal interplay mediated by** α**-synuclein?** 

elucidated.

organic molecules.

systems. Impairement of lysosomal and proteasomal protein degradation increases the burden of uncleared, unwanted proteins thus promoting their further accumulation and the development of a self-propagating cycle that eventually leads to cell death. Lysosomal function has been reported to decrease in PD patients (Alvarez-Erviti et al.; Chu, Dodiya, Aebischer, Olanow, & Kordower, 2009) and α-synuclein has been shown to be degraded by the lysosome specific mechanism of chaperone mediated autophagy (Cuervo, Stefanis, Fredenburg, Lansbury, & Sulzer, 2004; Xilouri et al., 2008).

Interestingly, Alvarez-Erviti et al. (2010), recently demonstrated that lysosomal inhibition in cells dramatically increased the intracellular and secreted pools of α-synuclein (Alvarez-Erviti et al.). The group further demonstrated a neuron-to-neuron exchange of cytosolic content via exosomes. It could be that under conditions which promote the intracellular accumulation of misfolded proteins, such as lysosomal and proteasomal dysfunction, the homeostatic mechanisms favor the secretion of aggregated forms of α-synuclein. Although the evidence for extracellular α-synuclein internalization in Emmanouilidou (2010) and Alvarez-Erviti (2010) studies are slightly debatable, there are strong indications at both that exosomes are an important mediator of intercellular communication. Exosome exchange between neurons might also represent a way for propagating pathological alterations throughout the brain during neurodegenerative diseases (Aguzzi & Rajendran, 2009; Smalheiser, 2007).

A demonstration that exosomes allow exchange of proteinaceaous or genetic material within the nervous system would provide an an explanation of how pathologies like Alzheimer's Creuzfeld Jacob or Parkinson's diseases, which begin in discrete regions spread overtime to connected regions of the central nervous system. This idea proposes that drugs directed toward reducing the formation and/or facilitating the clearance of misfolded α-synuclein, in order to arrest or reverse the self-propagation process, might represent novel therapeutic interventions for the treatment of PD. In addition, understanding how the neuropathology spreads throughout the nervous system in Parkinson's disease, will open up avenues for new treatments.

#### **3.4 Effects of extracellular** α**-synuclein on cellular homeostasis**

There are several studies addressing the role of extracellular α-synuclein especially in the context of PD pathology. The first indications that high levels of extracellular α-synuclein can impact cell viability came from studies using the recombinant protein. Exogenous addition of recombinant α-synuclein to the cultured medium of neuronal cells significantly decreased the viability of the recipient cells. Cell death was linearly correlated with the concentration of exogenous α-synuclein and was amplified when the applied protein also contained soluble oligomers (Albani et al., 2004; Du et al., 2003; Sung et al., 2001; Zhang et al., 2005). Application of recombinant monomeric or aggregated α-synuclein also revealed that this protein can be readily be uptaken by neuronal cells or even neural stem cells in culture (Ahn, Kim, Kang, Ryu, & Kim, 2006; Desplats et al., 2009; H. J. Lee et al., 2008; Luk et al., 2009; Sung et al., 2001). It has been suggested that the mechanism for α-synuclein internalization involves receptor-mediated endocytosis of the protein (Desplats et al., 2009; H. J. Lee et al., 2008; Sung et al., 2001). It has been proposed that this mechanism specifically mediates the uptake of oligomeric and fibrillar α-synuclein whereas monomeric α-synuclein enters cells via simple diffusion across the plasma membrane. Following internalization, extracellular α-synuclein was shown to move through the endosomal compartment and

Effects of Alpha-Synuclein on Cellular Homeostasis 183

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

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

KV and EE acknowledge support from the MJF Foundation and the EU 7th Framework

Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W. H., Castillo, P. E., et al.

dopamine system. *Neuron, 25*(1), 239-252.

(2000). Mice lacking alpha-synuclein display functional deficits in the nigrostriatal

target for the treatment of PD as well as other synucleinopathies.

**4. Conclusion** 

indeed very dynamic.

Program MEFOPA.

**6. References** 

**5. Acknowledgments** 

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 liposomes to assist the uptake.

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

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