Presynaptic Alpha-Synuclein Aggregation in a Mouse Model of Parkinson's Disease

Abstract

Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of ␣-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study ␣-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human ␣-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic ␣-synuclein existed primarily in an unbound, soluble pool. In contrast, ␣-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing ␣-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for ␣-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, ␣-synuclein aggregates are selectively found in presynaptic terminals.

Inhibition of Prolyl Oligopeptidase Restores Spontaneous Motor Behavior in the α-Synuclein Virus Vector–Based Parkinson's Disease Mouse Model by Decreasing α-Synuclein Oligomeric Species in Mouse Brain

“…This suggests that KYP-2047 mainly has an effect on soluble aSyn aggregates, with the PK-resistant fraction representing an oligomeric pool acquired before treatment initiation. Cremades et al (2012) showed that PK-resistant aSyn oligomers can be generated in initial stages at physiological concentrations, and PK-resistant species have been observed in presynaptic terminals in both transgenic animals (Tanji et al, 2010;Spinelli et al, 2014) and human disease with LBs, and they propose a mechanism that causes synaptic disturbances that are preceded by overt THϩ neuron loss (Kramer and SchulzSchaeffer, 2007). Our results suggest that AAV-aSyn injection caused an increase in both soluble and insoluble forms of oligomers, while evidence in the literature points to soluble aggregate forms being more toxic than insoluble aggregates (Karpinar et al, 2009;Winner et al, 2011;Rockenstein et al, 2014).…”

α-synuclein and synapsin III cooperatively regulate synaptic function in dopamine neurons

“…Along this line, our findings indicate that synapsin III is implicated in α-syn aggregation, although the molecular features of this effect warrant further investigation. To date, α-syn overexpression in transgenic mice has been found to associate with a decrease in synapsin-Ia/b, synapsinIIa/b and complexin-2 levels in whole brain extracts (Nemani et al, 2010), and its aggregation at cortical synapses induces a decrease of synapsin-I levels (Spinelli et al, 2014), but no study has determined whether α-syn is able to modulate synapsin III at present. Here, we have shown that α-syn interacts with and specifically modulates synapsin III in primary midbrain neurons.…”

Differential electrophysiological and morphological alterations of thalamostriatal and corticostriatal projections in the R6/2 mouse model of Huntington's disease

“…The tissue was then rinsed and left in 0.1 M phosphate buffer (PB) at 4°C until serially sectioned through the striatum (equivalent to +1.4 to +0.14 mm from Bregma) (Franklin and Paxinos, 2007) at 60 μm using a vibratome (Leica vibratome, Leica Microsystems). Pre-embed IHC of striatal tissue using DAB (Sigma, St Louis, MO) as the chromophore was performed as previously described (Spinelli et al, 2014) using antibodies against the DA D1 receptor (Sigma, St Louis, MO; polyclonal, 1:100 dilution), DA D2 receptor (Millipore, Burlington, MA; polyclonal, 1:50 dilution), VGLUT1 (Synaptic Systems, Gottingen, Germany; polyclonal 1:1000) and VGLUT2 (Synaptic Systems; polyclonal 1:100). Labeling for each antibody was carried out on separate pieces of striatal tissue, as well as double labeling for either the direct (D1) or indirect (D2) pathways, and VGLUT1 and VGLUT2.…”