The Stimulatory Effects of Intracellular α-Synuclein on Synaptic Transmission Are Attenuated by 2-Octahydroisoquinolin-2(1H)-ylethanamine.

α-Synuclein (αSyn) species can be detected in synaptic boutons, where they play a crucial role in the pathogenesis of Parkinson's Disease (PD). However, the effects of intracellular αSyn species on synaptic transmission have not been thoroughly studied. Here, using patch-clamp recordings in hippocampal neurons, we report that αSyn oligomers (αSynO), intracellularly delivered through the patch electrode, produced a fast and potent effect on synaptic transmission, causing a substantial increase in the frequency, amplitude and transferred charge of spontaneous synaptic currents. We also found an increase in the frequency of miniature synaptic currents, suggesting an effect located at the presynaptic site of the synapsis. Furthermore, our in silico approximation using docking analysis and molecular dynamics simulations showed an interaction between a previously described small anti-amyloid beta (Aß) molecule, termed M30 (2-octahydroisoquinolin-2(1H)-ylethanamine), with a central hydrophobic region of αSyn. In line with this finding, our empirical data aimed to obtain oligomerization states with thioflavin T (ThT) and Western blot (WB) indicated that M30 interfered with αSyn aggregation and decreased the formation of higher-molecular-weight species. Furthermore, the effect of αSynO on synaptic physiology was also antagonized by M30, resulting in a decrease in the frequency, amplitude, and charge transferred of synaptic currents. Overall, the present results show an excitatory effect of intracellular αSyn low molecular-weight species, not previously described, that are able to affect synaptic transmission, and the potential of a small neuroactive molecule to interfere with the aggregation process and the synaptic effect of αSyn, suggesting that M30 could be a potential therapeutic strategy for synucleinopathies.

Extracellular α-synuclein alters synaptic transmission in brain neurons by perforating the neuronal plasma membrane.

It has been postulated that the accumulation of extracellular α-synuclein (α-syn) might alter the neuronal membrane by formation of 'pore-like structures' that will lead to alterations in ionic homeostasis. However, this has never been demonstrated to occur in brain neuronal plasma membranes. In this study, we show that α-syn oligomers rapidly associate with hippocampal membranes in a punctate fashion, resulting in increased membrane conductance (5 fold over control) and the influx of both calcium and a fluorescent glucose analogue. The enhancement in intracellular calcium (1.7 fold over control) caused a large increase in the frequency of synaptic transmission (2.5 fold over control), calcium transients (3 fold over control), and synaptic vesicle release. Both primary hippocampal and dissociated nigral neurons showed rapid increases in membrane conductance by α-syn oligomers. In addition, we show here that α-syn caused synaptotoxic failure associated with a decrease in SV2, a membrane protein of synaptic vesicles associated with neurotransmitter release. In conclusion, extracellular α-syn oligomers facilitate the perforation of the neuronal plasma membrane, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We propose that α-synuclein (α-syn) oligomers form pore-like structures in the plasma membrane of neurons from central nervous system (CNS). We believe that extracellular α-syn oligomers facilitate the formation of α-syn membrane pore-like structures, thus explaining, in part, the synaptotoxicity observed in neurodegenerative diseases characterized by its extracellular accumulation. We think that alterations in ionic homeostasis and synaptic vesicular depletion are key steps that lead to synaptotoxicity promoted by α -syn membrane pore-like structures.

Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a cellular Alzheimer model.

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder and one of the current therapies involves strengthening the cholinergic tone in central synapses. Neuroprotective properties for nicotine have been described in AD, through its actions on nicotinic receptors and the further activation of the PI3K/Akt/Bcl-2 survival pathway. We have tested a quinolizidine alkaloid extract (TM0112) obtained from Teline monspessulanna (L.) K. Koch seeds to evaluate its action on nicotinic acetylcholine receptor (nAChR) in a neuronal AD model. Our data show that PC-12 cells pretreated with amyloid-ß (Aß) peptide for 24 h in presence of TM0112 modified Aß-reduction on cellular viability (Aß = 80 ± 3%; +TM0112 = 113 ± 4%, n = 15). In addition, this effect was blocked with atropine, MLA, and α-BTX (+TM0112+atropine = 87 ± 4%; +TM0112+MLA = 86 ± 4%; +TM0112+α-BTX = 92 ± 3%). Furthermore, similar protective effects were observed in rat cortical neurons (Aß = 63 ± 6%; +TM0112 = 114 ± 8%), but not in HEK293T cells (Aß = 61.4 ± 6.1%; +TM0112 = 62.8 ± 5.2) that do not express α7 nAChR. Moreover, the frequency of synaptic activity in the neuronal network (Aß = 51.6 ± 16.9%; +TM0112 = 210.8 ± 47.9%, n > 10), as well as the intracellular Ca2+ transients were recovered by TM0112 (Aß = 61.4 ± 6.9%; +TM0112 = 112.0 ± 5.7%; n = 3) in rat hippocampal neurons. TM0112 increased P-Akt, up to 250% with respect to control, and elevated Bcl-2/Bax percentage (Aß = 61.0 ± 8.2%; +TM0112 = 105.4 ± 19.5%, n = 4), suggesting a coupling between nAChR activation and an intracellular neuroprotective pathway. Our results suggest that TM0112 could be a new potential source for anti-AD drugs.