Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies.

Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol.

Biochemical fractionation of human α-Synuclein in a Drosophila model of synucleinopathies.

Synucleinopathies are a group of central nervous system pathologies that are characterized by neuronal accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in postmortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from brains of aged flies. We also assessed the effect of sonication on solubility of human α-synuclein and optimized a protocol to discriminate relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the 3-step protocol distinguishes between cytosolic soluble proteins in fraction 1, detergent-soluble proteins in fraction 2 and insoluble proteins in fraction 3. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and enriching fraction 2 of the protocol.

Reduced Plasma-Membrane Calcium ATPase Activity and Extracellular Acidification Trigger Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to an increase in neurotransmitter release that restores the strength of synaptic transmission following a blockade of nicotinic acetylcholine receptors (nAChRs). Mechanisms informing the presynaptic terminal of the loss of postsynaptic receptivity remain poorly understood. Previous research at the mouse NMJ suggests that extracellular protons may function as a retrograde signal that triggers an upregulation of neurotransmitter output (measured by quantal content, QC) through the activation of acid-sensing ion channels (ASICs). We further investigated the pH-dependency of PHP in an ex-vivo mouse muscle preparation. We observed that increasing the buffering capacity of the perfusion saline with HEPES abolishes PHP and that acidifying the saline from pH 7.4 to pH 7.2-7.1 increases QC, demonstrating the necessity and sufficiency of extracellular acidification for PHP. We then sought to uncover how the blockade of nAChRs leads to the pH decrease. Plasma-membrane calcium ATPase (PMCA), a calcium-proton antiporter, is known to alkalize the synaptic cleft following neurotransmission in a calcium-dependent manner. We hypothesize that since nAChR blockade reduces postsynaptic calcium entry, it also reduces the alkalizing activity of the PMCA, thereby causing acidosis, ASIC activation, and QC upregulation. In line with this hypothesis, we found that pharmacological inhibition of the PMCA with carboxyeosin induces QC upregulation and that this effect requires functional ASICs. We also demonstrated that muscles pre-treated with carboxyeosin fail to generate PHP. These findings suggest that reduced PMCA activity causes presynaptic homeostatic potentiation by activating ASICs at the mouse NMJ.