Αξιολόγηση της επίδρασης μεταβλημένων επιπέδων ντοπαμίνης σε μοντέλα διαγονιδιακών ποντικών που φέρουν τον παθολογικό φαινότυπο Α-συνουκλεΐνης A53T

Abstract

Parkinson’s disease and other synucleinopathies share two key features, neuron denegeration and insoluble protein aggregates consinsting mainly of α-synuclein. Aggregation of α-synuclein is considered a pathogenic process and therefore has been studied extensively. Dopamine and its metabolic products are shown to modulate the polymerization process of α-synuclein in vitro. In order to study this interaction in vivo we delivered to the substantia nigra of transgenic mice that over-express the disease-associated mutant of α-synuclein, A53T, a lentivirus carrying the coding sequence of tyrosine hydroxylase (TH). Transduction with a mutant form of TH, that lacks dopamine feedback inhibition, led to increased concentration of striatal dopamine. Even though the age of onset and presentation of phenotype are not changed due to dopamine increase, biochemical analysis has verified accumulation of distinct triton-soluble higher molecular weight α-synuclein species in the nigra. Analysis for selective cell death and presence of α-synuclein positive inclusions within the nigra did not indicate altered pathology. The results of this study indicate that targeted increase of dopamine in the nigral neurons profoundly altered α-synuclein aggregation however it did not correlate to increased neurotoxicity.Despite the potential importance of the oligomeric α-synuclein intermediates of the aggregation process in neuron function, their biochemical and pathobiological relevance in vivo remains vastly unknown. Here we employed two-dimensional analytical separation and an array of biochemical and cell based assays to characterize α-synuclein oligomers that are present in the nervous system of A53T α-synuclein transgenic mice. The most prominent species identified were 53 Å detergent soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing α-synuclein inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea, but were sensitive to proteinase-K digestion. Even though the oligomers shared similar basic biochemical properties, those obtained from inclusion bearing regions were prominently reactive to antibodies that recognize oxidized α-synuclein oligomers, significantly accelerated aggregation of α-synuclein in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion bearing regions were not toxic and delayed the in vitro formation of α-synuclein fibrils. These data indicate that specific conformations of α-synuclein oligomers are present in distinct brain regions of A53T α-synuclein transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties, but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region specific intrinsic factors.