Kisspeptin-10 Mitigates α-Synuclein-Mediated Mitochondrial Apoptosis in SH-SY5Y-Derived Neurons via a Kisspeptin Receptor-Independent Manner

The hypothalamic neurohormone kisspeptin-10 (KP-10) was inherently implicated in cholinergic pathologies when aberrant fluctuations of expression patterns and receptor densities were discerned in neurodegenerative micromilieus. That said, despite variable degrees of functional redundancy, KP-10, which is biologically governed by its cognate G-protein-coupled receptor, GPR54, attenuated the progressive demise of α-synuclein (α-syn)-rich cholinergic-like neurons. Under explicitly modeled environments, in silico algorithms further rationalized the surface complementarities between KP-10 and α-syn when KP-10 was unambiguously accommodated in the C-terminal binding pockets of α-syn. Indeed, the neuroprotective relevance of KP-10’s binding mechanisms can be insinuated in the amelioration of α-syn-mediated neurotoxicity; yet it is obscure whether these extenuative circumstances are contingent upon prior GPR54 activation. Herein, choline acetyltransferase (ChAT)-positive SH-SY5Y neurons were engineered ad hoc to transiently overexpress human wild-type or E46K mutant α-syn while the mitigation of α-syn-induced neuronal death was ascertained via flow cytometric and immunocytochemical quantification. Recapitulating the specificity observed on cell viability, exogenously administered KP-10 (0.1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated apoptosis and mitochondrial depolarization in cholinergic differentiated neurons. In particular, co-administrations with a GPR54 antagonist, kisspeptin-234 (KP-234), failed to abrogate the robust neuroprotection elicited by KP-10, thereby signifying a GPR54 dispensable mechanism of action. Consistent with these observations, KP-10 treatment further diminished α-syn and ChAT immunoreactivity in neurons overexpressing wild-type and E46K mutant α-syn. Overall, these findings lend additional credence to the previous notion that KP-10’s binding zone may harness efficacious moieties of neuroprotective intent.


Introduction
Pervasive dissemination of α-synuclein (α-syn)-laden inclusions in the immediate vicinity of cholinergic neurons exemplifies the defining neuropathological phenotype of dementia with Lewy Bodies (DLB) [1,2]. The sporadic manifestations of these anomalous deposits, albeit paralleled by aberrant increments in wild-type α-syn expression [3], have been causally linked to familial pedigrees bearing the E46K mutation [4]. Indeed, irrespective of whether these lesions are themselves pathogenic, their omnipresence reflects the functional compromise of α-syn, a presynaptically-localized 140 amino acid protein [5]. While the biological purpose of α-syn remains indeterminate, at supraphysiological levels, α-syn may be extruded in a stochastic manner from an initiating subpopulation of selectively vulnerable neurons [6]. The consequence is a myriad of pathogenically variant deposits, each of which entails the premature accretion of α-syn in characteristic patterns and locations [7][8][9]. transiently transfected α-syn constructs on cholinergic differentiated neurons were consecutively analyzed by means of an annexin-V-affinity assay. Herein, we report that significant increases in apoptotic demise were discerned amongst neurons overexpressing human wild-type (59.3 ± 1.2%, *** p < 0.001) and E46K mutant α-syn (61.4 ± 2.2%, *** p < 0.001) compared with the GFP-positive neurons. In an attempt to further delineate the neuroprotective relevance of KP-10/GPR54 signaling in α-syn-induced apoptosis, transfected neurons were exogenously treated with KP-10 (0.1 µM) either alone or in concert with KP-234 (0.1 to 10 µM). Intriguingly, while the apoptosis-dependent externalization of phosphatidylserine by both human wild-type and mutant α-syn was substantially suppressed by KP-10 (*** p < 0.001), co-administrations with the GPR54 antagonist KP-234 had a negligible influence on this ameliorative effect (Figure 1a-d).
neuroprotective relevance of KP-10/GPR54 signaling in α-syn-induced apoptosis, transfected neurons were exogenously treated with KP-10 (0.1 µM) either alone or in concert with KP-234 (0.1 to 10 µM). Intriguingly, while the apoptosis-dependent externalization of phosphatidylserine by both human wild-type and mutant α-syn was substantially suppressed by KP-10 (*** p < 0.001), co-administrations with the GPR54 antagonist KP-234 had a negligible influence on this ameliorative effect (Figure 1a-d). The quantification of total apoptotic cells presented in (b,d) is representative of mean values ± SEM from three independent biological replicates performed in triplicates. While the externalization of plasma membrane phosphatidylserine by both human wild-type (a,b) and E46K mutant (c,d) αsyn was considerably inhibited by KP-10 (*** p < 0.001), co-administrations with KP-234 had no discernible impact on this ameliorative effect. Statistical significance was defined as p-value less than 0.001 (*** p < 0.001).

KP-10 Rescues α-Syn-Induced Mitochondrial Depolarization in Cholinergic-like Neurons through a GPR54 Dispensable Mechanism
Working on the premise that α-syn-mediated lethality may, in part, necessitate the instigation of the intrinsic apoptotic cascade, mitochondrial integrity of α-syn-transfected neurons was subsequently probed by tracking the intramitochondrial sequestration of a    ChAT immunoreactions revealed significant increases of fluorescence intensity in both human wildtype (*** p < 0.001) and E46K mutant α-syn (*** p < 0.001) expressing neurons compared with the GFP-labeled neurons. This α-syn-induced upsurge in immunostaining intensity was considerably diminished by the neuroprotective effects of KP-10 (*** p < 0.001). Statistical significance was defined as p-value less than 0.001 (*** p < 0.001).

Discussion
Aberrant expressions of human wild-type and E46K mutant α-syn across cholinergic phenotypes have been causally linked to pathogenically noxious inclusions in the perturbed DLB microenvironment [34,35]. The widespread dissemination of these deleterious deposits, irrespective of whether it emanates intracellularly or extracellularly, may sensitize compromised neurons to spontaneous mitochondrial depolarizations, thereby rendering them more predisposed to apoptogenic consequences [11,36]. Since prophylactically intervening this commensurate loss of neurons has yet to result in disease-modifying therapeutics, there is an unmet requisite to counteract α-syn-centric extra-and intracellular pathogenic cascades. We have previously showcased that exogenous KP-10 was efficacious in diminishing the progressive demise of α-syn-rich ChAT-positive neurons with preferential docking affinities, hence reflecting a noncanonical extracellular binding interaction [22]. The recent revelation that GPR54 is endowed with a neuroprotective potential further buttressed the conjecture that KP-10 may engender a formerly unattainable level of pharmacologically-actionable specificity by additionally exploiting intracellular pro-survival signaling cascades [30]. Thus, with (a) Differentiated neurons overexpressing human wild-type or E46K mutant α-syn were doubleimmunostained with anti-α-syn (green) and anti-ChAT (red) antibodies, co-stained with Dapi (blue), and representative images were subsequently taken before and after treatment with KP-10. Scale bars, 50 µm. In this context, 60 neurons were analyzed per experiment. The staining intensity and quantification of α-syn and ChAT expression levels presented in (b) are representative of mean values ± SEM from three independent biological experiments. Quantitative analysis of α-syn and ChAT immunoreactions revealed significant increases of fluorescence intensity in both human wildtype (*** p < 0.001) and E46K mutant α-syn (*** p < 0.001) expressing neurons compared with the GFP-labeled neurons. This α-syn-induced upsurge in immunostaining intensity was considerably diminished by the neuroprotective effects of KP-10 (*** p < 0.001). Statistical significance was defined as p-value less than 0.001 (*** p < 0.001).

Discussion
Aberrant expressions of human wild-type and E46K mutant α-syn across cholinergic phenotypes have been causally linked to pathogenically noxious inclusions in the perturbed DLB microenvironment [34,35]. The widespread dissemination of these deleterious deposits, irrespective of whether it emanates intracellularly or extracellularly, may sensitize compromised neurons to spontaneous mitochondrial depolarizations, thereby rendering them more predisposed to apoptogenic consequences [11,36]. Since prophylactically intervening this commensurate loss of neurons has yet to result in disease-modifying therapeutics, there is an unmet requisite to counteract α-syn-centric extra-and intracellular pathogenic cascades. We have previously showcased that exogenous KP-10 was efficacious in diminishing the progressive demise of α-syn-rich ChAT-positive neurons with preferential docking affinities, hence reflecting a non-canonical extracellular binding interaction [22]. The recent revelation that GPR54 is endowed with a neuroprotective potential further buttressed the conjecture that KP-10 may engender a formerly unattainable level of pharmacologically-actionable specificity by additionally exploiting intracellular pro-survival signaling cascades [30]. Thus, with these premises in mind, cholinergic-like neurons were transiently engineered to overexpress human wild-type or E46K mutant α-syn, while KP-10 s mitigation of α-syn-induced neuronal death was probed by virtue of GPR54 antagonism. Herein, we report that neurons overexpressing human wild-type and E46K mutant α-syn were equally susceptible to apoptotic demise and temporally coincided with the dissipation of the mitochondrial transmembrane potential. Reiterating the specificity we observed preliminarily [22], exogenously administered KP-10 (0.1 µM) further repressed α-syn-mediated apoptosis and mitochondrial depolarization in cholinergic differentiated neurons. In particular, co-administrations with the GPR54 antagonist, kisspeptin-234, failed to nullify the robust neuroprotection elicited by KP-10, thereby signifying a GPR54 dispensable mechanism of action. Consistent with these findings, KP-10 treatment simultaneously diminished α-syn and ChAT expression levels in neurons overexpressing the human wild-type and E46K mutant α-syn. Indeed, if one were to extrapolate the results obtained from the above, it is conceivable that the preferential vulnerability of ChAT-positive neurons to mitochondrial apoptosis emanates from α-syn's inherent propensity to modulate ChAT expression levels. The likelihood that this postulate has some merit has risen a notch or two [13,33], and it becomes reasonable to enquire as to how KP-10 restored ChAT expression to near-physiologic levels. As a seemingly reliable marker of anomalous cholinergic transmission [37,38], the reduction in ChAT expression is the most widely scrutinized facet of cholinergic pathology [39]. Nonetheless, spatial and temporal decreases in ChAT levels do not necessarily reflect cognitive deterioration considering that its theoretical capacity to synthesize acetylcholine is far in excess of the actual rate of acetylcholine biosynthesis [38,40]. Instead, paradoxical upregulation of ChAT expression has been ascertained in prodromal states, thus insinuating that such counterintuitive manifestations may compensate in unique chemoplastic fashions upon interactions with α-syn-enriched lesions [14]. As such, for neurotherapeutic initiatives to be efficacious, they must first harbor the latent ability to foster intrinsic and extrinsic pathways of neuronal integrity [41,42]. In this sense, we have identified KP-10 as a neuroprotectant that averts intrinsic apoptosis by modulating the externalization of plasma membrane phosphatidylserine and permeabilization of mitochondrial membranes. Mechanistically, the concerted action of GPR54 signaling kinases that operate either upstream or downstream of the aforementioned events is believed to account for whether neurons survive or succumb to α-syn's noxious insults. The manner by which these downstream effectors are selectively governed, however, remains ill-defined since the pharmacological blockade of GPR54 had no discernible influence on KP-10 s salutary effects. At first consideration, the possibility of an inadequate receptor blockade cannot be entirely precluded, given that a certain degree of disparity in terms of antagonistic potency has been apparent under various experimental conditions [43][44][45][46]. Alternatively, the basis for such a phenomenon could also be indicative of a bona fide neuroprotective maneuver since these observations appear to resonate with our previously established in silico models, which subserved transient KP-10-α-syn complexations [22]. While the dynamical details of these explicitly modeled "adducts" provided more ambiguity on KP-10 s non-classical functional versatility, such dichotomies in modes of action were crucial in redirecting aggregation-predisposed α-syn into offpathway non-toxic accretions [47]. Intriguingly, tertiary interactions with the C-terminal residues of α-syn were able to safeguard the C-terminus from bimolecular self-assembly and steer the expansion of a loop that is no longer primed for the generation of β-sheet-rich aggregates [48][49][50][51][52][53][54]. Though not exhaustive, there seems to be a basis for the apparent redundancy of GPR54 signaling in SH-SY5Y-derived neurons, for ideally, an insurmountable antagonist should considerably annul the intrinsic activity of an agonist [43,46]. Guided by this notion, the systematic substitution of amino acid residues along the core of KP-10 initially established a consensus sequence for antagonism that retained high-affinity receptor engagement but failed to elicit signaling in GPR54-expressing neurons [44]. To reconcile with the pleiotropic nature of its analog, KP-234 carries a substitution of Tyr 1 with D-Ala and of Ser 5 with Gly, with further enrichment by the replacement of Leu 8 with D-Trp [44]. The fact that Tyr 1 and Ser 5 appear to constitute a binding pharmacophore that must be retained to effectuate KP-10-α-syn interactions [22] imparts credence to the notion that KP-234 s binding zone may not be able to harness functional moieties of protective relevance. With continued expansions in large-scale co-immunoprecipitations amidst GPR54-ablated neurons, established means of direct protein-protein interactions between KP-10 and α-syn will be essential for reconciling our initial theoretical predictions. Undeniably, the structural commonalities between KP-10 and α-syn made it tempting to propose that it is the extracellular interaction between these two proteins that drove the concerted attenuation of α-syn and ChAT expression levels. This hypothetical model, which is tentatively depicted in Figure 4, integrates the remodeling of toxic fibrils into non-toxic aggregates, yet whether the nature of this direct-binding interaction is confounded by the intricately woven proteostatic burden α-syn creates, warrants further clarification. While it may be of heuristic value to causally insinuate KP-10 s functionality in this regard, immunofluorescence analyses of α-syn clearance using autophagy-specific and aggregation state-specific antibodies may shed light on the KP-10-centric "fail-safe" mechanisms that partake in modulating α-syn proteostasis and expression levels.
Ser 5 with Gly, with further enrichment by the replacement of Leu 8 with D-Trp [44]. The fact that Tyr 1 and Ser 5 appear to constitute a binding pharmacophore that must be retained to effectuate KP-10-α-syn interactions [22] imparts credence to the notion that KP-234′s binding zone may not be able to harness functional moieties of protective relevance. With continued expansions in large-scale co-immunoprecipitations amidst GPR54-ablated neurons, established means of direct protein-protein interactions between KP-10 and αsyn will be essential for reconciling our initial theoretical predictions. Undeniably, the structural commonalities between KP-10 and α-syn made it tempting to propose that it is the extracellular interaction between these two proteins that drove the concerted attenuation of α-syn and ChAT expression levels. This hypothetical model, which is tentatively depicted in Figure 4, integrates the remodeling of toxic fibrils into non-toxic aggregates, yet whether the nature of this direct-binding interaction is confounded by the intricately woven proteostatic burden α-syn creates, warrants further clarification. While it may be of heuristic value to causally insinuate KP-10′s functionality in this regard, immunofluorescence analyses of α-syn clearance using autophagy-specific and aggregation state-specific antibodies may shed light on the KP-10-centric "fail-safe" mechanisms that partake in modulating α-syn proteostasis and expression levels.

Cell Culture
The

Differentiation into Cholinergic-like Neurons
SH-SY5Y-derived cholinergic-like neurons were generated using a differentiation methodology adapted from our previously documented protocol [22]. Concisely, SH-SY5Y cells were appropriately cultured in DMEM encompassing 0.5% FBS and 10 µM of all-trans retinoic acid (RA) (Sigma-Aldrich, St Louis, MO, USA; Cat. #: R2625) for 72 h to prompt neuronal 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. To that end, cells were never sub-cultured beyond passage 20 in order to circumvent any distinct phenotypic alteration in differentiated cholinergic cells.

Transient Transfections
The transient transfection of plasmid DNA in RA-differentiated cholinergic neurons was accomplished by employing Lipofectamine 3000 reagents (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: L3000001). Briefly, 2.5 µg of pEGFP-C1, 2.5 µg of pcDNA6 wild-type α-syn, or 2.5 µg of pcDNA6 E46K mutant α-syn plasmids were diluted in 125 µL of Opti-MEM serum-reduced medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: 31985070) and 5 µL of P3000 reagent, respectively. Succeeding a 5 min incubation step at room temperature (RT), 7.5 µL of Lipofectamine 3000 reagent was diluted in 125 µL of Opti-MEM serum-reduced medium, added into the diluted Opti-MEM-DNA solution, and incubated for an additional 15 min to assemble DNA-Lipofectamine 3000 lipoplexes. The complex concoctions were thereafter added dropwise into each well of a 6-well plate and re-incubated in a humidified 5% CO 2 incubator for 2-4 days. The estimation of transfection efficiency in RA-differentiated cholinergic neurons was achieved by enumerating the amount of GFP-positive neurons under a Nikon Eclipse 90i fluorescent microscope (Nikon, Melville, NY, USA).

Quantification of Apoptosis by Annexin-V Labeling
Quantitative analysis of apoptotic profile in α-syn-overexpressing cholinergic cells was carried out using the Muse™ Annexin-V & Dead Cell Assay Kit (Luminex, Austin, TX, USA; Cat. #: MCH100105) in accordance with the manufacturer's guidelines. Briefly, the assay utilizes fluorescently labeled annexin-V in combination with a dead cell marker, 7-Aminoactinomycin D (7-AAD), to detect the translocation of phosphatidylserine to the external membrane of apoptotic cells. SH-SY5Y cells were first seeded into 6-well plates (NEST Biotechnology, Wuxi, China; Cat. #: 703001) at a density of 2.5 × 10 5 cells/well and cultured overnight. Following RA-induced differentiation and transient transfection of plasmids, cells were treated with KP-10 (0.1 µM) alone or in combination with KP-234 (0.1 to 10 µM) for 24 h in 5% CO 2 at 37 • C. In this context, the selection of kisspeptin doses was based on previously published concentration-response curves and our very own preliminary observations [22]. Upon incubation, cells were harvested, gently rinsed with ice-cold PBS, collected by centrifugation at 1000 rpm for 5 min, and re-suspended in DMEM containing 1% FBS. The cell suspensions were then stained with 100 µL of Muse™ Annexin-V & Dead Cell Reagent and incubated for 20 min at RT in the dark. The events for live, early apoptotic, late apoptotic, and necrotic cells were finally analyzed by the Muse™ Cell Analyzer (Luminex, Austin, TX, USA).

Mitochondrial Membrane Potential
Mitochondrial membrane depolarization in α-syn-overexpressing cholinergic cells was evaluated using the Muse™ MitoPotential Kit (Luminex, Austin, TX, USA; Cat. #: MCH100110) according to the manufacturer's specifications. SH-SY5Y cells were initially plated into 6-well plates (NEST Biotechnology, Wuxi, China; Cat. #: 703001) at a density of 2.5 x 10 5 cells/well and cultivated overnight. Upon RA-mediated differentiation and transient transfection of plasmids, cells were treated with KP-10 (0.1 µM) alone or in concert with KP-234 (0.1 to 10 µM) at 37 • C for 24 h in a 5% CO 2 incubator. Following incubation, cells were harvested, centrifuged at 1000 rpm for 5 min, and re-suspended in 1X assay buffer. The Muse MitoPotential working solution was subsequently prepared by diluting the MitoPotential dye in 1X assay buffer (1:1000). Accordingly, 100 µL of cell suspension was added to 95 µL of MitoPotential working solution and incubated at 37 • C for 20 min. Eventually, 5 µL of 7-AAD reagent was methodically added, mixed thoroughly, and incubated for 5 min at RT. Lastly, changes in mitochondrial potential were determined by the Muse ® Cell Analyzer (Luminex, Austin, TX, USA).

Statistical Analysis
The experimental results were unveiled as mean ± SE from three independent biological experiments. Statistical analysis was appraised via one-way analysis of variance (ANOVA) prior to Tukey's post hoc tests for all multiple assessments (IBM SPSS Statistics v24). A p-value of less than 0.05 was defined as a statistically significant difference.