Single point mutations at the S129 residue of α-synuclein and their effect on structure, aggregation, and neurotoxicity.

Parkinson's disease is an age-related neurological disorder, and the pathology of the disease is linked to different types of aggregates of α-synuclein or alpha-synuclein (aS), which is an intrinsically disordered protein. The C-terminal domain (residues 96-140) of the protein is highly fluctuating and possesses random/disordered coil conformation. Thus, the region plays a significant role in the protein's solubility and stability by an interaction with other parts of the protein. In the current investigation, we examined the structure and aggregation behavior of two artificial single point mutations at a C-terminal residue at position 129 that represent a serine residue in the wild-type human aS (wt aS). Circular Dichroism (CD) and Raman spectroscopy were performed to analyse the secondary structure of the mutated proteins and compare it to the wt aS. Thioflavin T assay and atomic force microscopy imaging helped in understanding the aggregation kinetics and type of aggregates formed. Finally, the cytotoxicity assay gave an idea about the toxicity of the aggregates formed at different stages of incubation due to mutations. Compared to wt aS, the mutants S129A and S129W imparted structural stability and showed enhanced propensity toward the α-helical secondary structure. CD analysis showed proclivity of the mutant proteins toward α-helical conformation. The enhancement of α-helical propensity lengthened the lag phase of fibril formation. The growth rate of ß-sheet-rich fibrillation was also reduced. Cytotoxicity tests on SH-SY5Y neuronal cell lines established that the S129A and S129W mutants and their aggregates were potentially less toxic than wt aS. The average survivability rate was ∼40% for cells treated with oligomers (presumably formed after 24 h of incubation of the freshly prepared monomeric protein solution) produced from wt aS and ∼80% for cells treated with oligomers obtained from mutant proteins. The relative structural stability with α-helical propensity of the mutants could be a plausible reason for their slow rate of oligomerization and fibrillation, and this was also the possible reason for reduced toxicity to neuronal cells.

Small-Molecule Cdc25A Inhibitors Protect Neuronal Cells from Death Evoked by NGF Deprivation and 6-Hydroxydopamine.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that are presently incurable. There have been reports of aberrant activation of cell cycle pathways in neurodegenerative diseases. Previously, we have found that Cdc25A is activated in models of neurodegenerative diseases, including AD and PD. In the present study, we have synthesized a small library of molecules targeting Cdc25A and tested their neuroprotective potential in cellular models of neurodegeneration. The Buchwald reaction and amide coupling were crucial steps in synthesizing the Cdc25A-targeting molecules. Several of these small-molecule inhibitors significantly prevented neuronal cell death induced by nerve growth factor (NGF) deprivation as well as 6-hydroxydopamine (6-OHDA) treatment. Lack of NGF signaling leads to neuron death during development and has been associated with AD pathogenesis. The NGF receptor TrkA has been reported to be downregulated at the early stages of AD, and its reduction is linked to cognitive failure. 6-OHDA, a PD mimic, is a highly oxidizable dopamine analogue that can be taken up by the dopamine transporters in catecholaminergic neurons and can induce cell death by reactive oxygen species (ROS) generation. Some of our newly synthesized molecules inhibit Cdc25A phosphatase activity, block loss of mitochondrial activity, and inhibit caspase-3 activation caused by NGF deprivation and 6-OHDA. Hence, it may be proposed that Cdc25A inhibition could be a therapeutic possibility for neurodegenerative diseases and these Cdc25A inhibitors could be effective treatments for AD and PD.