Abstract
Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.
Highlights
Neurodegeneration in Parkinson’s disease (PD) is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).[3]
The same effect was seen in TH-negative neurons where overexpression of all αSyn variants resulted in a significant reduction of the mean single neurite length compared with controls (EGFP: 53 ± 1.2 μm; αSyn-WT: 42 ± 1.8 μm; αSyn-A30P: 45 ± 1.4 μm; αSyn-A53T: 47 ± 1.8 μm)
In order to establish whether differences in autophagy are due to altered binding of αSyn to autophagosomes, we evaluated the colocalization of αSyn and light chain 3 (LC3) in transfected PMN by confocal microscopy (Figure 5)
Summary
Neurodegeneration in PD is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).[3]. Wild-type (WT) human αSyn does not lead to major clinical or histological abnormalities when expressed in transgenic mice,[11,12] but its overexpression mediated by adeno-associated viral vectors (AAV) results in severe neurodegeneration, suggesting a dose-dependent toxic effect.[13,14] Different human αSyn-A30P and -A53T transgenic mouse lines develop severe motor impairments, partly resembling symptoms of human PD, accompanied by Received 17.11.14; revised 14.5.15; accepted 15.5.15; Edited by D Bano a degeneration of the nigrostriatal neuronal system and LBlike pathology.[11,12,15] In line with the pathological findings in human PD, the axonal compartment is affected early and most prominently in these animal models. Several studies have shown that the inhibition of autophagy results in an accumulation and increased toxicity of αSyn, whereas the activation of autophagy has therapeutic effects in PD models.[23,24,25,26] the direct effects of αSyn and its mutants on autophagy seem to rely strongly on the model system and the published data are highly controversial.[24,26,27,28,29,30,31,32]
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