How does loss of presynaptic α-syn contribute to neurodegeneration?
The scope of our one subject per treatment condition pilot study in non-human primates has clear limitations. Nevertheless, the results confirm our previous findings in the rat (Gorbatyuk et al., 2010; Kanaan and Manfredsson, 2012). At this stage our findings, while internally consistent among the treatment conditions, must be considered as generating a hypothesis, rather than being conclusive. While the idea that maintaining α-syn expression is critical for survival of populations of adult neurons has received relatively little investigation, the high prevalence and widespread distribution of α-syn in brain inarguably reflects an important function in synapses. While direct evidence for the loss-of-function hypothesis is in its early stages, many studies provide indirect support for the concept.
Alpha-synuclein exhibits a dramatic redistribution within the neuronal compartment as a function of aging: the best-validated primary risk factor for PD. A quantitative morphological postmortem analysis of α-syn immunoreactivity within SN DA neurons of humans reveals a distinct pattern of increased staining within neuronal soma with advancing chronological age (Chu and Kordower, 2007). Samples were from individuals with a mean age of 31 years (young group, N = 6), 55 years (middle-aged, N = 4) and 84 years (aged, N = 8). Alpha-synuclein immunoreactivity was rarely detected in the soma of SN neurons in young individuals and was restricted to its normal location in the neuropil. With advancing age, a progressive increase in somatic immunoreactivity was observed, culminating in a 639% increase in aged subjects. Quantitation of the intensity of somatic staining on a per neuron basis confirmed this pattern, illustrating a mean 57% increase in aged subjects as compared to young. It is noteworthy that none of these samples exhibited α-syn aggregates, suggesting that this intracellular redistribution of α-syn may be an ongoing aging-related event that precedes processes of aggregation. The same pattern was observed in aging monkeys. Of importance to the loss-of-function hypothesis, no comparison to α-syn distribution/levels in striatum was performed, providing no opportunity to assess whether increased somatic levels are associated with decreased synaptic levels.
Oxidative stress is an established threat to DA neuron viability and can be significantly exacerbated by DA mishandling (i.e., increased cytosolic DA). Indeed, multiple lines of evidence suggest a regulatory role for synuclein in dopamine neurotransmission (Perez et al., 2002; Maries et al., 2003; Wersinger et al., 2003), consistent with the view that loss of presynaptic α-syn may contribute to increased oxidative stress via dysregulation of DA biosynthesis, release and metabolism. Studies in primary cultures of DA neurons demonstrate that in conditions of elevated oxidative stress, endogenous levels of α-syn increase and provide neuroprotection (Quilty et al., 2006; Musgrove et al., 2011). Similarly, transgenic mice over-expressing α-syn are resistant to paraquat-induced toxicity for DA neurons known to be associated with oxidative stress (Manning-Bog et al., 2003). Thus, endogenous α-syn likely provides an important DA neuron defense against oxidative damage.
Mishandling of synaptic DA and the toxic byproducts of its metabolism in the cytosol have a long history of association with the vulnerability of these neurons (e.g., Perez and Hastings, 2004; Segura-Aguilar et al., 2014). Studies of transgenic mice deficient in α-syn, while free of overt DA neuron pathology in adulthood, exhibit changes in DA neurotransmission, with some of these becoming exaggerated in aged animals. Adult, triple knock-out mice for all synuclein family proteins exhibit elevated evoked release of DA in striatum, enhanced turnover and reduced presynaptic DA stores (Anwar et al., 2011). Study of two α-syn deficient mouse lines found evidence for increased stimulated DA overflow in striatum, higher basal extracellular DA levels, decreased expression of the dopamine transporter (DAT) and reduced DA reuptake (Chadchankar et al., 2011). Aged (24–26 months old) α-syn null mice exhibit significant reduction of striatal DA, a decrease in TH+ fibers and decreased striatal levels of TH and DAT (Al-Wandi et al., 2010). These data support the view that α-syn is a pivotal presynaptic regulator of DA neurotransmission and that disruption of this process may result in chronic accumulation of DA in the cytosol.
Perhaps the most direct evidence for an α-syn loss-of-function process related to aggregation comes from the mouse pre-formed fibril (PFF) model of synucleinopathy (Luk et al., 2012). In this model, intracerebral injection of pre-formed fibrils of α-syn seeds misfolding of endogenous α-syn, resulting in progressive accumulation of cytoplasmic aggregates of the protein. In a recent report (Osterberg et al., 2015) the formation of aggregates was tracked over time. The findings indicate that as inclusions mature into a compact form, detection of soluble α-syn shows a parallel decline.