Towards Uncovering the Role of Pre-fibrillar Oligomers of á-Synuclein in the Pathogenesis of Parkinson's Disease

Parkinson s disease (PD) is a progressive neuropathological disorder that is characterized by the presence of intra-cytoplasmic inclusions called Lewy Bodies (LBs). LBs represent insoluble protein aggregates with á-Synuclein (áS) as a major component. LBs also mark lesions found in a range of related disorders, collectively referred to as synucleinopathies. Recent studies suggest that pathogenesis could result from small pre-fibrillar intermediates of the aggregation pathway, i.e. soluble oligomers of misfolded á-Synuclein. So, it is not decided yet whether the soluble oligomers or the final products of the áS aggregation (i.e. the áS fibrils) are cause of the disease, meaning that the nature of pathogenic áS species is still unresolved. To understand the nature of pathogenic áS species and to investigate whether accumulation of soluble pre-fibrillar oligomers of áS is responsible for the increased neurotoxicity in PD, I have used rationally designed structural variants of áS with different aggregation properties and tested their biological properties in Drosophila melanogaster, which lacks an áS gene in the wild type genome. Two of the designed variants, TP-áS and A56P-áS, have a remarkably decreased propensity to aggregate, and they form more pre-fibrillar soluble oligomeric species in aggregation assays in vitro. In order to examine their biological effects in vivo, I have employed Drosophila as a tool to explore possible PD-like effects of the áS mutants. The áS mutant genes were inserted into the Drosophila genome. To avoid possible position effects affecting the expression of the individual transgenes, I used the phiC31 site-specific integration system. The site specific genomic integration of the áS gene and the different áS mutants permit to directly compare the effects of the different áS variants on various biological parameters such as longevity, climbing (negative geotactic and phototactic responses), sleep behavior and circadian rhythmicity that were used as sensitive readouts for neurotoxicity in response to áS activity expressed specifically in the Dopaminergic (DA) neurons. The results show that the TP-áS and A56P-áS mutants caused increased neurotoxicity as compared to wild type áS or expression of the bacterial lacZ gene serving as controls. Immunohistochemistry on adult fly brains confirm that neurotoxicity as revealed by the behavior tests correlates well with the degeneration of DA neurons in response to the expression of the áS variants. The data show that the pre-fibrillar oligomer-forming TP-áS mutant is most toxic among the áS variants. The results are consistent with the conclusion that pre-fibrillar oligomers of áS are high ly toxic pathogenic species in the neurodegeneration process associated with PD. Aside from supporting the hypothesis that the pre-fibrillar status of áS is already toxic for neurons, the Drosophila system presented here delivers a novel experimental system in which the non-motor aspects of PD, the mechanism of cellular action and likely even the efficacy of lead compounds leading to therapeutic drugs can be addressed

Pre-Fibrillar α-Synuclein Mutants Cause Parkinson's Disease-Like Non-Motor Symptoms in Drosophila

Parkinson's disease (PD) is linked to the formation of insoluble fibrillar aggregates of the presynaptic protein α-Synuclein (αS) in neurons. The appearance of such aggregates coincides with severe motor deficits in human patients. These deficits are often preceded by non-motor symptoms such as sleep-related problems in the patients. PD-like motor deficits can be recapitulated in model organisms such as Drosophila melanogaster when αS is pan-neurally expressed. Interestingly, both these deficits are more severe when αS mutants with reduced aggregation properties are expressed in flies. This indicates that that αS aggregation is not the primary cause of the PD-like motor symptoms. Here we describe a model for PD in Drosophila which utilizes the targeted expression of αS mutants in a subset of dopadecarboxylase expressing serotonergic and dopaminergic (DA) neurons. Our results show that targeted expression of pre-fibrillar αS mutants not only recapitulates PD-like motor symptoms but also the preceding non-motor symptoms such as an abnormal sleep-like behavior, altered locomotor activity and abnormal circadian periodicity. Further, the results suggest that the observed non-motor symptoms in flies are caused by an early impairment of neuronal functions rather than by the loss of neurons due to cell death

Age-dependent circadian defects in response to wild type αS, TP-αS, EKO/Kir2.1 and NaChBac expression in DA neurons.

<p>(<b>A</b>, <b>B</b>) Double-plotted actograms of young flies (3 days after hatching) expressing wild type αS (WT-αS) (A) and TP-αS (B) under the control of the DA neuron specific <i>TH-Gal4</i> driver. T refers to circadian periodicity which is 23.8 hrs in both cases. (<b>C</b>, <b>D</b>) Double-plotted actograms of old flies (30 days after hatching) expressing WT-αS and TP-αS. Note the different circadian periodicity in response to WT-αS (T = 23.7 hrs) and TP-αS expression (T = 26.7 hrs). (<b>E</b>, <b>F</b>) Double-plotted actogram of old flies expressing EKO/Kir2.1 (E) or NaChBac (F) under the control of the DA neuron specific <i>TH-Gal4</i> driver. Note the similar extension of the circadian periodicities (T = 27.6 hrs and T = 27.0 hrs, respectively) as observed after TP-αS expression. All experiments (n = 32–58 flies) were carried out under constant dark conditions after the animals were kept in a dark-light cycle of 12∶12 hrs. T was calculated by the Chi-squared periodogram analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024701#s4" target="_blank">Materials and Methods</a>).</p

Locomotor activity and anticipation of the dark-light transition of flies expressing <i>lacZ</i>, wild type αS and the oligomer-forming TP-αS mutant.

<p>(<b>A</b>) Dark-light (D∶L = 12 hrs∶12 hrs) transition bar. (<b>B–G</b>) Locomotor activity profile of young flies (3 days after hatching; <b>B</b>, <b>D</b>, <b>F</b>) and old flies (30 days after hatching; <b>C</b>, <b>E</b>, <b>G</b>); expressing <i>lacZ</i> (blue line; <b>B</b>, <b>C</b>), WT-αS (green line; <b>D</b>, <b>E</b>) and TP-αS (red line; <b>F</b>, <b>G</b>). Black arrows point to the beginning of locomotor activity prior to the onset of light (anticipatory behavior of the flies). Note that old flies expressing TP-αS fail to anticipate the onset of the light period (red arrow in <b>F</b>). Red asterisks show the phasing out of the maximum locomotor activities after the light-dark switch. For details see text.</p

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α-synuclein (αS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure-based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function