Intra-neuronal aggregates of α-synuclein known as Lewy bodies or Lewy neurites are key neuropathological alterations in multiple brain regions in patients with Parkinson's disease (PD). Interestingly, Lewy pathology is also evident in the enteric nervous system in PD patients and in some neurologically normal individuals with constipation (Bloch et al. 2006; Shannon et al. 2012). Braak and colleagues (Braak et al. 2006) proposed that α-synuclein pathology is present in the periphery before it reaches the brain and triggers the motor symptoms of PD. They went on to suggest that it spreads along interconnected neural pathways as early as 10–20 years before the onset of any motor symptoms. Recent studies have suggested that release of cellular α-synuclein into the extracellular space might contribute to the spreading of α-synuclein aggregates between neurons. Thus, experimental evidence in cell culture systems indicates that α-synuclein is indeed released by many neuronal cells (Fig. 1). In the study by ‘Paillusson et al.’ in this issue, the authors provide a novel perspective on the secretion of α-synuclein in primary cultures of enteric neurons and show that this is activation dependent, which is in contrast to secretion mechanisms reported by others (Paillusson et al. 2012).
Earlier observations of α-synuclein secretion, in various cell culture systems, indicated that it is secreted via a non-classical pathway, independent of a stimulus (Fig. 1). In α-synuclein transfected SH-SY5Y cells, the release mechanism has been shown to involve α-synuclein-containing exosomal vesicles, secreted by a brefeldin A-independent exocytosis pathway (Emmanouilidou et al. 2010; Alvarez-Erviti et al. 2011; Danzer et al. 2012). Various forms of cellular stress have been reported to further enhance the translocation of intracellular α-synuclein into vesicles, resulting in increased release (Jang et al. 2010). In addition, this release appears to be Ca2+-dependent, but independent of oxidative stress or selective protease inhibitors (Emmanouilidou et al. 2010). In these studies, the secreted α-synuclein was detected as both monomeric and oligomeric species. Culture medium containing these forms of α-synuclein was toxic when added to naive SH-SY5Y cells. Congo Red and scyllo-inositol, which are compounds that bind and possibly disrupt amyloid structures, inhibited the neurotoxic effects of the secreted α-synuclein (Emmanouilidou et al. 2010). In summary, earlier studies have shown that some neuroblastoma cells can release aggregated α-synuclein through ER/Golgi-independent, non-classical exocytosis in vesicular structures to the surrounding medium.
The highlighted study of Paillusson et al. applies a different approach and is one of few studies to focus on primary neuronal cells, instead of immortalized neuroblastoma lines like transfected SH-SY5Y. Thus, the authors extend this previously published work, demonstrating that primary cultures of enteric neurons are capable to express and secrete α-synuclein (Paillusson et al. 2010). They now show for the first time that primary cultures of enteric neurons secrete α-synuclein via classic exocytosis. This proceeds through endoplasmic reticulum/Golgi related transport and can be blocked by application of the endoplasmic reticulum inhibitor brefeldin-A (Fig. 1). The data implicate a direct exocytosis; the secreted form of α-synuclein appears to be vesicle independent. They detect the α-synuclein by a detergent-free and specific ELISA, further supporting the notion that the released α-synuclein is free, or at least not sterically hindered by a vesicular membrane.
As described above, in transfected SH-SY5Y cells and primary neuronal cells, α-synuclein exocytosis is predominantly executed by a non-classical exocytosis pathway in a vesicle-associated form (Fig. 1). The vesicular release of aggregated, truncated, and post-translationally modified α-synuclein forms via exosomes might be because of cellular quality control mechanisms disposing of accumulated or damaged proteins (Emmanouilidou et al. 2010; Alvarez-Erviti et al. 2011; Danzer et al. 2012). This hypothesis is supported by the fact that increased levels of intracellular α-synuclein parallel augmented release of extracellular α-synuclein-containing vesicles.
In contrast, Paillusson et al. now report that secretion of α-synuclein by primary enteric neurons is increased upon Na+-channel mediated depolarization, which triggers the classic protein release pathway of cells (Fig. 1). Moreover, they found that augmented α-synuclein expression by enteric neurons did not influence or correlate with extracellular α-synuclein levels, which points to a highly regulated release mechanism. Concomitant with the regulated secretion of α-synuclein in the culture medium, the authors reported no cytotoxic effect. This result might be explained by three different potentially connected circumstances: (i) differences in the active form of secreted α-synuclein, (ii) the free release of the protein into the medium occurring independently of vesicular transport, or (iii) the phagocytic potential of glia cells co-cultured in the medium. The missing cytotoxic effect of α-synuclein released by primary enteric neurons is at odds with the published data generated in transfected SH-SY5Y cells and allows for speculation whether there are indeed various forms of secreted α-synuclein and whether some may have a physiological function or serve as a paracrine signal. For instance, under physiological conditions enteric neurons are in intimate contact with the gut environment and exposed to pathogens. These environmental factors might initiate cell activation and concomitant release of α-synuclein which in turn could trigger a local innate immune response. It remains to be elucidated to which extent this fraction of secreted α-synuclein results in pathological aggregation and age-related propagation to neuroanatomically connected organs and ultimately to the brain.
To unravel the role of peripheral α-synuclein in the onset and spreading of Lewy body-like pathology, experimental models have been essential to understand α-synuclein release into the extracellular space and uptake by neighboring cells. Interneuronal transmission of extracellular α-synuclein has been suggested to occur in patients therapeutically grafted with neuronal stem cells (Brundin et al. 2008) and several mechanisms, aiming to explain the underlying cell-to-cell propagation, have been proposed. Mouse cortical neural stem cells either co-cultured with neurons overexpressing human α-synuclein or grafted into transgenic mice expressing human α-synuclein can rapidly develop inclusions of human α-synuclein (Desplats et al. 2009). In addition, over-expression of a mutant form of dynamin1, which blocks endocytosis, resulted in reduced intercellular transmission of α-synuclein, which was released by co-cultured transfected SH-SY5Y cells (Desplats et al. 2009). These results point to vesicle-derived uptake by endocytosis as one of the mechanisms of cell-to-cell propagation. Linked to the mechanism of α-synuclein transmission, the molecular structure of secreted α-synuclein is likely a crucial factor determining seeding of intracellular α-synuclein inclusions and subsequent death of the cell taking up the α-synuclein. Thus, oligomeric and fibrillary α-synuclein structures, rather than a monomeric form, induce α-synuclein deposition in the recipient neuron (Luk et al. 2009).
The study by Paillusson et al. raises fundamental questions concerning what extracellular forms of α-synuclein can induce pathological processes (Paillusson et al. 2012). Is release of α-synuclein from neuronal cells per se driving protein aggregation, neuroinflammation, and neuronal degeneration, and if so are only oligomers and fibrils driving these events? Which other pathways are involved in the secretion of α-synuclein and do they differ between neuronal cell types of different organs? Could organ-specific activation of neurons in the enteric nervous system (ENS) and the central nervous system (CNS) result in an altered life cycle of α-synuclein regulated differentially by expression, degradation, and clearance? How is extracellular α-synuclein taken up by cells and in which way does it induce intracellular pathological deposits? Designing the right experiments to these questions is of particular importance to elucidate the potential peripheral onset of PD.
One viable approach might be to turn to human specimens. By screening patient material, Braak and colleagues identified intra-neuronal α-synuclein deposits in the olfactory bulb, in the enteric nervous system and in other peripheral ganglia projecting to the CNS (Braak et al. 2003). Their pioneering work allowed them to postulate a potential onset of PD in the olfactory system and the gut followed by a propagation of the disease to the CNS, including the substantia nigra in the midbrain (Braak et al. 2006; Hawkes et al. 2007). Such a dual-hit hypothesis could explain the appearance of some non-motor symptoms in patients (e.g., constipation and anosmia), which precede the onset of CNS-related motor symptoms (Braak et al. 2006). Interestingly, this hypothesis is supported by recent results from mice which developed α-synuclein deposits in the ENS upon oral application of the pesticide rotenone. Although still awaiting confirmation by an independent group, the observed progressive appearance of α–synuclein deposits along neuroanatomically linked systems implies a propagation of the pathology along the gut-brain axis (Pan-Montojo et al. 2010).
Paillusson et al. do not mention if α-synuclein is taken up by co-cultured glia cells. Enteric neurons have peripheral macrophages as immediate neighbors, representing the first line of defense in the gut. Peripheral macrophages actively screen their environment to attack and phagocytize infectious agents thereby releasing immune and neuro-modulatory factors (Lema Tome et al. 2012). The constantly hostile environment represented by the contents of the gut may explain why enteric neurons in the gut wall might exhibit a high risk to become activated and to release α-synuclein. Similarly, this environment may also render enteric neurons more susceptible to attack by toxic forms of α-synuclein.
The findings of Paillusson et al. and the effects of the local gut on ENS-specific secretion pathways for α-synuclein could help to address if the Braak hypothesis of disease onset in the gut is valid. If the amount of extracellular α-synuclein in the gut wall innervated by the ENS could be linked to disease onset or severity stage, ENS-specific forms of secreted α-synuclein might serve as biomarkers for synucleinopathies and enable early diagnosis of the disease by regular colonic biopsies (Shannon et al. 2012). In addition, if enteric α-synuclein aggregation and constipation precede PD, early detection could open a window for therapeutic intervention before the appearance of motor symptoms. Therapeutic approaches might foster on selective inhibition of the pathway controlling enteric α-synuclein secretion or on neutralizing the secreted form of α-synuclein.
In conclusion, Paillusson and colleagues introduced a new and attractive concept of α-synuclein release by cells derived from primary peripheral neurons. Future work should focus on the fate of extracellular α-synuclein derived from enteric neurons. This will help to clarify if extracellular α-synuclein derived from the ENS plays an important normal physiological function or could be a trigger of pathology.