Abstract
α-Synuclein is the major pathological component of synucleinopathies including Parkinson's disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.
Highlights
Introduction aSynuclein is one of the major components of Lewy bodies (LBs) and Lewy neurites (LNs), which are pathological hallmarks of synucleinopathies including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) [1,2,3]
To characterize the neurons with low expression of a-synuclein, we performed immunostaining using a specific antibody against glutamic acid decarboxylase (GAD), a well-known marker of inhibitory neurons
GAD-positive puncta were juxtaposed with the a-synucleinpositive dendrites, most GAD signals were not colocalized with a-synuclein (Fig. 1A, merged images). 8961.4% of GAD-positive neurons displayed no expression of a-synuclein (6 coverslips were analyzed)
Summary
Introduction aSynuclein is one of the major components of Lewy bodies (LBs) and Lewy neurites (LNs), which are pathological hallmarks of synucleinopathies including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) [1,2,3]. Insights into the mechanisms underlying a-synuclein pathology are crucial for the understanding of these neurodegenerative disorders. To elucidate the mechanisms of neuronal loss in these diseases, it is important to determine the physiological roles of a-synuclein in normal neurons. A-Synuclein is localized at presynapses in vivo and in vitro [7,8,9]. It has been suggested that a-synuclein is involved in the generation and maintenance of synapses, because this protein appears earlier than synaptophysin during development of the central nervous system and is localized to presynaptic terminals throughout the adult mammalian brain [10,11]. A-Synuclein directly binds to synaptobrevin-2 in presynaptic regions and functions to sustain soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly in vivo and in vitro [12]. In a recent study using an overexpression and knockout model in vitro, it was reported that a-synuclein maintains the size of the vesicular recycling pool [13]
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