Kisspeptin-10 Rescues Cholinergic Differentiated SHSY-5Y Cells from α-Synuclein-Induced Toxicity In Vitro

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn’s non-amyloid-β component (NAC) and Aβ’s C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn’s deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments.


Introduction
Dementia with Lewy bodies (DLB) is an idiopathic neurodegenerative entity, pathologically lesioned by conspicuous degeneration of α-synuclein (α-syn)-rich cholinergic neurons in the senile brain [1][2][3]. Across pathological subtypes, the α-syn gene was incriminated in cholinergic neurons when the point mutation, E46K, was ascertained in kindreds with mixed phenotypes of parkinsonism and dementia that resembled DLB [4]. Beyond this pathogenetic continuum, co-immunoprecipitation experiments from human brain extracts further theorized a pervasive scenario for DLB pathogenesis encompassing heterologous collusion between α-syn and the amyloid-β (Aβ) peptide [5,6]. A 14-kDa monomer ubiquitously expressed in presynaptic cholinergic termini; the α-syn is inherently Scale bars, 100 μm. The percentage of ChAT-positive neurons is representative of mean values ± SEM from three independent biological replicates. More than 95% of RA-treated SHSY-5Y cells established a cholinergic-like phenotype by displaying ChAT-like immunoreactivity at culture day three.

Low-Dose Exposure to KP-10 has No Effect on RA-Differentiated Cholinergic Cell Viability
To verify that KP-10 by itself has no modulatory effect on cellular proliferation or cytotoxicity that may obscure its neuroprotective potential against α-syn-induced toxicity, differentiated SHSY-5Y neurons were first exposed to various doses of KP-10 and assayed for 3-(4, 5-dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide (MTT) uptake. Analysis of cell viability following a 24-h treatment showed that KP-10 in the concentration range between 0.01 to 5 μM has a negligible influence on RA-differentiated cholinergic cells. By contrast, treatment of cultured cells with 10 µM of KP-10 significantly reduced cell survival down to 80% after 24 h (** p < 0.01) ( Figure 2).   Scale bars, 100 μm. The percentage of ChAT-positive neurons is representative of mean values ± SEM from three independent biological replicates. More than 95% of RA-treated SHSY-5Y cells established a cholinergic-like phenotype by displaying ChAT-like immunoreactivity at culture day three.

Low-Dose Exposure to KP-10 has No Effect on RA-Differentiated Cholinergic Cell Viability
To verify that KP-10 by itself has no modulatory effect on cellular proliferation or cytotoxicity that may obscure its neuroprotective potential against α-syn-induced toxicity, differentiated SHSY-5Y neurons were first exposed to various doses of KP-10 and assayed for 3-(4, 5-dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide (MTT) uptake. Analysis of cell viability following a 24-h treatment showed that KP-10 in the concentration range between 0.01 to 5 μM has a negligible influence on RA-differentiated cholinergic cells. By contrast, treatment of cultured cells with 10 µM of KP-10 significantly reduced cell survival down to 80% after 24 h (** p < 0.01) ( Figure 2). Cholinergic differentiated SH-SY5Y cells were exposed to KP-10 in the concentration range between 0.01 to 10 µM for 24 h. The data presented in Figure 2 are representative of mean values ± SEM from three independent biological replicates performed in triplicates. While low doses of KP- Cholinergic differentiated SH-SY5Y cells were exposed to KP-10 in the concentration range between 0.01 to 10 µM for 24 h. The data presented in Figure 2 are representative of mean values ± SEM from three independent biological replicates performed in triplicates. While low doses of KP-10 (0.01-5 µM) displayed no significant differences in cellular viability, 10 µM of KP-10 decreased cell survival down to 80%. Statistical significance was defined as p value less than 0.01 (** p < 0.01).

KP-10 Binds to the C-Terminal Residues of Wild-Type and E46K Mutant α-Syn In Silico
The intermolecular binding interactions between KP-10 and human wild-type or E46K mutant α-syn were initially probed recursively based upon CDOCKER interaction energies (-kcal/mol). Relying on binding site topology and intermolecular affinity, a lower CDOCKER interaction energy implies greater binding affinity. According to this parameter, KP-10 binds favorably to wild-type α-syn with a CDOCKER energy score of −118.049 kcal/mol. Detailed evaluation of docked poses further revealed that key amino acid residues of KP-10 (112-117) generated hydrogen bonds with Glu126, Glu130, Glu131, Gly132, and Asp135 of wild-type α-syn. Intriguingly, the binding mode assessment of KP-10 with E46K mutant α-syn displayed analogous binding patterns to the same binding site, generating hydrogen bonds with Glu126, Glu130, and Asp135 of E46K mutant with a docking score of −114.869 kcal/mol. The docked conformations of KP-10 with human wild-type or E46K mutant α-syn, along with key interactions between putative binding site residues, are illustrated in Figure 6a,b. In this context, although in silico predictions rationalized the surface complementarities between KP-10 and both α-syns in terms of docking affinities, biological constraints such as protein flexibility and solvation dynamics were not fully considered. Thus, to compensate for the aforementioned limitations, MD simulations were consecutively executed to comprehend the time-dependent stability of KP-10-α-syn complexes in an explicit solvent environment. Based on the simulation's trajectories, the conformational landscape of docked complexes was subsequently parameterized according to root-mean-square deviation (RMSD) values. Herein, we report that KP-10 was unambiguously accommodated spatially in the C-terminal binding pockets of both α-syns throughout the course of the simulation. While the wild-type and E46K mutant α-syn mechanically unfolded into relatively linear configurations, with their respective conformational flexibilities increasing monotonically towards the very C-terminus, bound KP-10 molecules stabilized after 200 trajectory frames. The MD trajectories of docked KP-10-α-syn complexes, along with their corresponding RMSD values, are depicted in Figure 7a,b and Supplementary Videos S1 and S2. mutant α-syn-induced toxicity was markedly suppressed by 0.01, 0.1, 1, 5 (*** p < 0.001) and 10 µM (** p < 0.01) of KP-10 (b). Statistical significance was defined as p value less than 0.01 and 0.001 (** p < 0.01 and *** p < 0.001).

KP-10 Binds to the C-terminal Residues of Wild-Type and E46K Mutant α-Syn In Silico
The intermolecular binding interactions between KP-10 and human wild-type or E46K mutant α-syn were initially probed recursively based upon CDOCKER interaction energies (-kcal/mol). Relying on binding site topology and intermolecular affinity, a lower CDOCKER interaction energy implies greater binding affinity. According to this parameter, KP-10 binds favorably to wild-type α-syn with a CDOCKER energy score of −118.049 kcal/mol. Detailed evaluation of docked poses further revealed that key amino acid residues of KP-10 (112-117) generated hydrogen bonds with Glu126, Glu130, Glu131, Gly132, and Asp135 of wild-type α-syn. Intriguingly, the binding mode assessment of KP-10 with E46K mutant α-syn displayed analogous binding patterns to the same binding site, generating hydrogen bonds with Glu126, Glu130, and Asp135 of E46K mutant with a docking score of −114.869 kcal/mol. The docked conformations of KP-10 with human wild-type or E46K mutant α-syn, along with key interactions between putative binding site residues, are illustrated in Figure 6a,b. In this context, although in silico predictions rationalized the surface complementarities between KP-10 and both α-syns in terms of docking affinities, biological constraints such as protein flexibility and solvation dynamics were not fully considered. Thus, to compensate for the aforementioned limitations, MD simulations were consecutively executed to comprehend the time-dependent stability of KP-10-α-syn complexes in an explicit solvent environment. Based on the simulation's trajectories, the conformational landscape of docked complexes was subsequently parameterized according to root-mean-square deviation (RMSD) values. Herein, we report that KP-10 was unambiguously accommodated spatially in the C-terminal binding pockets of both α-syns throughout the course of the simulation. While the wild-type and E46K mutant α-syn mechanically unfolded into relatively linear configurations, with their respective conformational flexibilities increasing monotonically towards the very C-terminus, bound KP-10 molecules stabilized after 200 trajectory frames. The MD trajectories of docked KP-10-αsyn complexes, along with their corresponding RMSD values, are depicted in Figure 7a,b and Supplementary Videos S1 and S2.

Discussion
ChAT-positive SH-SY5Y neurons were first engineered ad hoc to transiently overexpress human wild-type or E46K mutant α-syn, while α-syn-mediated cytotoxicity was subsequently quantified using an MTT-based metabolic assay that determines neuronal survival. Cumulatively, overexpression of the pathological mutation E46K exhibited elevated α-syn mRNA levels, which correlated more distinctly with the degree of cellular toxicity as compared to its wild-type counterpart in cholinergic neuronal-like cells. Our results, thereby, cohere with the hypothesis that a pronounced upsurge of α-syn mRNA expression over baseline levels could have detrimental consequences against cholinergic neurons, efficiently mimicking the neuropathology of DLB. The up-regulation of human wild-type and E46K mutant α-syn as a result of aberrant gene dosage has consistently augmented the formation of extracellular aggregation-prone morphologies that yielded neuronal toxicity when overexpressed in cellular models [33][34][35]. Given that α-syn-laden cholinergic neurons are a prerequisite for exacerbated DLB pathology [36][37][38], various approaches have been employed to abrogate the toxic insults of aggregated α-syn by counteracting extra-and intracellular pathogenic pathways [39][40][41]. Such approaches range from the extracellular enhancement of chaperone-based therapeutics that refold aberrant α-syn conformations [16] to strategies aimed at inhibiting intracellular beta-pleated α-syn aggregates [39,42]. As such, since the NAC fragment of human α-syn binds preferentially to Aβ and facilitates its abnormal aggregation [10], it is tempting to speculate that compounds which specifically bind to extracellular α-syn or Aβ may be neuroprotective [22,43]. To address this multi-hit hypothesis, we then explored whether exogenous administration of KP-10 might play a critical role in the attenuation of neuronal cell death induced by α-syn in ChAT-positive neurons. Upon treatment with exogenous KP-10, while a high concentration of KP-10 (10 µM) unexpectedly decreased the cellular viability of RA-differentiated cholinergic cells, low concentrations of KP-10 (0.01-1 µM) significantly suppressed wild-type and E46K mutant α-syn-induced toxicity. In marked contrast, however, an altered dose-response curve was observed in cholinergic differentiated cells following low dose exposure to KP-10 (0.01-0.1 µM), with an apparent decrease in neuroprotective efficacy when in the presence of wild-type or E46K mutant α-syn. This discrepancy in response variability is indicative of a possibility that α-syn may be compromising the intracellular transduction of kisspeptin-GPR54 signaling in RA-differentiated cholinergic cells. More sophisticated receptor binding studies may shed light on this possibility, but such a phenomenon might only be demonstrable under dynamic conditions in which cellular responses are investigated via a GPR54 antagonist. Consistent with the in vitro outcomes are theoretical predictions from molecular docking algorithms, which demonstrated that KP-10 binds favorably to specific sites of wild-type and E46K mutant α-syn with CDOCKER interaction energies of −118.049 kcal/mol and −114.869 kcal/mol, respectively. In particular, key amino acid residues of KP-10 (112-117) were found to have generated hydrogen bonds with the C-terminal residues (Glu126, Glu130, Glu131, Gly132, and Asp135) of α-syn. A visual inspection of MD trajectories over a 50 ns time-scale further suggested that the KP-10-α-syn complexes were relatively stable despite solvent-driven perturbations. Throughout the span of the simulation, KP-10 re-docked into the C-terminus of both α-syns upon disassembly, thus reflecting the existence of a putative binding site. Although the mechanistic details of these "stable adducts" are somewhat arbitrary, the elucidation of these findings may insinuate otherwise. It became apparent from early in vitro experimentations that transient long-range tertiary interactions between the N and C termini of α-syn are crucial for spontaneous NAC-mediated aggregation [44][45][46][47][48]. Contrary to expectations, naturally existing compounds that bind to α-syn with binding sites at both N-and C-termini have been shown to redirect aggregation-prone α-syn into off-pathway non-toxic assemblies [49][50][51][52]. Intriguingly, the α-syn folds back onto itself to form a loop that inhibits α-syn aggregation since the NAC fragment is no longer primed to create a linear conformation required for the formation of β-sheet-rich aggregates [44,53]. Guided by this notion, natural molecules that bind to the C-terminal residues of α-syn were able to shield the C-terminus from bimolecular self-assembly and preclude intramolecular contacts between the N and C termini, thereby impeding misfolding and ensuing aggregation [54][55][56][57]. Taking these observations into consideration, it seems plausible that the 6-residue fragment (112-117) of KP-10 could be specifically binding to the C-terminus of α-syn by mediating a physical influence that attenuates the likelihood of misfolding to confer neuroprotection in vitro. The fact that the exact peptide fragment was able to bind predominantly to extracellular Aβs to promote neuronal survival [22] provides credence to the notion that KP-10's binding zone may harness functional moieties of neuroprotective significance. We thus conclude from these complementary investigations that the neuroprotective relevance of KP-10 binding mechanisms can be structurally and functionally implicated in α-syn-mediated toxicity of cholinergic neuronal-like cells. Stemming from this purported facet, further exploration into KP-10's mode of action may provide the basis for novel structure-based drug design of α-syn-interfering aggregation inhibitors. Indeed, the identification of this non-canonical binding interface endows KP-10 with the ability to engender a previously unattainable level of pharmacological selectivity by targeting not just the α-syn but specific pro-survival signaling cascades. However, as it probably represents a biologically relevant interaction with the kisspeptin-GPR54 signaling pathway, subsequent biophysical characterizations by virtue of GPR54 antagonism are warranted.

Differentiation of SHSY-5Y into Cholinergic Neurons
Cholinergic neuronal-like cells were generated using a differentiation procedure modified from a previously described protocol [30]. Briefly, SHSY-5Y cells were plated in DMEM containing 0.5% FBS and 10 µM of all-trans retinoic acid (RA) (Sigma-Aldrich, St Louis, MO, USA; Cat. #: R2625) for 72 h to induce cholinergic differentiation. After three days in the presence of all-trans RA, the differentiation medium was replenished with DMEM containing 10% FBS for the transient transfection of plasmids. In general, cells were used between passages 17 to 19 and were never passaged beyond passage 19 in order to avoid any potential phenotypic change in RA-differentiated cholinergic cells.

Plasmid Transfections
The   Finally, the MTT-containing medium was carefully siphoned out, and 150 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA; Cat. #: D8418) was added to each well to solubilize the formazan crystals that had formed. The absorbance of each well was measured at 570 nm with a reference wavelength of 650 nm on a microplate reader (TECAN Infinite M200 Pro, Männedorf, Switzerland). The background light scattering at 650 nm was then subtracted from the values obtained for formazan absorbance (570 nm).

RNA Isolation and cDNA Synthesis
The RNA isolation of α-syn-transfected RA-differentiated cholinergic cells was performed by first dissolving the cells in 200 µL of Tri-RNA reagent (Sigma-Aldrich, St Louis, MO, USA; Cat. #: T9424). Chloroform was then added in a 1:5 ratio and shaken to mix thoroughly, followed by centrifugation at 12,000 g, 4 • C for 15 min. The aqueous phases were carefully removed into fresh centrifuge tubes, and isopropyl alcohol was then added at a 1:2 ratio. The mixtures were next incubated at RT for 10 min, followed by centrifugation at 12,000× g, 4 • C for 15 min. The supernatants produced were consequently discarded while the pellets were rinsed with 75% ethanol, followed by centrifugation at 7500 g, 4 • C for 5 min. The pellets were then air-dried for 5 min and subsequently dissolved in 30 µL of Milli-Q water. The purity of the RNAs extracted was determined using the nanodrop-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA isolations (initial input amounts of 1000 ng) with a 260/280 value of above 1.90 were deemed as pure and reverse transcribed into cDNAs using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA; Cat. #: 4374966).

In Silico Molecular Docking
The X-ray crystal structure of human α-syn was first retrieved from the Protein Data Bank (www.rcsb.org; Accessed on 21 May 2021) with a PDB ID of 1XQ8. KP-10 was next constructed in ChemDraw Professional 15.0 with the peptide sequence of H-YNWNSFGLRF-[NH2], identical to the ones used in vitro. All computational-based analyses were eventually carried out using the docking software CDOCKER with BIOVIA Discovery Studio 4.0 (San Diego, CA, USA). CDOCKER is an algorithm that employs CHARMm force fields to adopt a basic strategy involving the generation of several initial ligand orientations in the active site of target proteins followed by molecular dynamics-based simulated annealing and a final refinement via energy minimization [59]. Accordingly, α-syn and KP-10 proteins were energy-minimized using the CHARMm force field. The binding site of α-syn was then retrieved from the 'receptor cavities' of the software and utilized for docking purposes. KP-10 was consequently docked into the probable binding site of α-syn to obtain the best possible conformations, which are built upon total docking energy. The pose or conformation having the highest dock score was deemed as having the most favorable interaction. This procedure was first performed on the wild-type α-syn and subsequently repeated for the E46K mutant. For the virtual mutation of wild-type α-syn, the 'Build Mutant' protocol was used for the substitution of amino acid residues. Here, using wild-type α-syn as a template, the E46K mutant was generated by substituting glutamic acid with lysine on the protein sequence. Energy minimization for the optimization of residue geometry was then performed on the E46K mutant using the algorithm of smart minimization until gradient tolerance (RMS Gradient 0.1 kcal/mol/A • ) was satisfied. KP-10 was ultimately docked into the same binding site as in the case of the wild-type α-syn.

Explicit-Solvent MD Simulations
To parameterize the dynamic range of KP-10-α-syn complexes in an explicit solvent, MD simulations were carried out within the YASARA graphical user interface using the Amber 14 force field [60]. Briefly, all MD simulations utilized an explicit solvation system described by the TIP3P water model and a cubic periodic boundary that extended 20 Å around the KP-10-α-syn complexes. Electrostatic interactions were predicated by the Particle mesh Ewald (PME) method for long-range coulombic forces. The entire system was energy-minimized with 5000 steps of steepest descent followed by 5000 steps of conjugate gradients to eradicate conformational stress. In order to mimic physiological conditions, KP-10-α-syn interactions were solvated with water molecules and subsequently neutralized with counter ions with 0.9% NaCl salt at 303 K temperature. The temperature of the simulation system was governed by the Berendsen thermostat, and the final production phase of simulations was successively carried out for a total of 50 ns MD simulations. Finally, the trajectory frames were calculated with a time step of 1.25 fs, and the simulation's snapshots were captured every 100 ps. MD simulations for both wild-type and E46K mutant α-syn complexes were probed under identical experimental conditions.

Statistical Analysis
Data were presented as mean ± SE from three independent biological experiments. Statistical analysis was evaluated using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests for all multiple comparisons (IBM SPSS Statistics v24). A p-value of less than 0.05 was defined as a statistically significant difference.