Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT7 Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

Jakubowska, Klaudia; Hogendorf, Adam S.; Gołda, Sławomir; Jantas, Danuta

doi:10.1007/s11064-024-04159-z

Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT₇ Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

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Published: 04 June 2024

Volume 49, pages 2179–2196, (2024)
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Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT₇ Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

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Abstract

There is some evidence that the serotonin receptor subtype 7 (5-HT₇) could be new therapeutic target for neuroprotection. The aim of this study was to compare the neuroprotective and neurite outgrowth potential of new 5-HT₇ receptor agonists (AH-494, AGH-238, AGH-194) with 5-CT (5-carboxyamidotryptamine) in human neuroblastoma SH-SY5Y cells. The results revealed that 5-HT₇ mRNA expression was significantly higher in retinoic acid (RA)-differentiated cells when compared to undifferentiated ones and it was higher in cell cultured in neuroblastoma experimental medium (DMEM) compared to those placed in neuronal (NB) medium. Furthermore, the safety profile of compounds was favorable for all tested compounds at concentration used for neuroprotection evaluation (up to 1 μM), whereas at higher concentrations (above 10 μM) the one of the tested compounds, AGH-194 appeared to be cytotoxic. While we observed relatively modest protective effects of 5-CT and AH-494 in UN-SH-SY5Y cells cultured in DMEM, in UN-SH-SY5Y cells cultured in NB medium we found a significant reduction of H₂O₂-evoked cell damage by all tested 5-HT₇ agonists. However, 5-HT₇-mediated neuroprotection was not associated with inhibition of caspase-3 activity and was not observed in RA-SH-SY5Y cells exposed to H₂O₂. Furthermore, none of the tested 5-HT₇ agonists altered the damage induced by 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium ion (MPP +) and doxorubicin (Dox) in UN- and RA-SH-SY5Y cells cultured in NB. Finally we showed a stimulating effect of AH-494 and AGH-194 on neurite outgrowth. The obtained results provide insight into neuroprotective and neurite outgrowth potential of new 5-HT₇ agonists.

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Introduction

Neurodegenerative diseases (ND) are becoming an increasingly serious medical and economical issue [1]. Lack of available clinical treatments and high prevalence of ND like Alzheimer’s disease, Parkinson’s disease (PD), Huntington’s disease, multiple sclerosis and amyotrophic lateral sclerosis creates an urgent need for therapies which could attenuate or stop the disease progression [2, 3]. Although the etiology of ND differs, pathomechanisms like mitochondrial dysfunction, neuroinflammation, excitotoxicity, excessive oxidative stress and dysregulation of cell death pathways are common among them [4,5,6,7]. Despite years of research, the scientist are still trying to discover potential therapeutic targets. In this context 5-HT₇ (serotonin type 7 receptor) seems to be an interesting target for novel therapies. 5-HT₇ belongs to rhodopsin-like transmembrane G-protein coupled receptors (GPCRs) and was discovered as the last one from the family of serotonin receptors in 1993 [8]. It is widely expressed throughout the whole brain with the highest expression found in thalamus, hypothalamus, hippocampus and amygdala [9, 10]. The mRNA for this receptor was present also in raphe nucleus, caudate nucleus, putamen as well as in substantia nigra. Moreover, neurons, as well as astrocytes and microglia express this receptor [11]. All the splice variants of this serotonin receptor, coupled with G_s protein connected with stimulation of cAMP production, show high level of constitutive activity [12, 13]. By acting on G_s protein, adenyl cyclase (AC) is activated which results in rapid increase of intercellular cAMP level. This secondary messenger acts on protein kinase A (PKA) and causes phosphorylation of downstream effector proteins like extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) [14, 15]. The signaling pathways activated by stimulation of 5-HT₇ like protein kinase A (PKA), extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) play a crucial role in maintaining proper neuron function, support the establishment of neural networks and mediate neuroprotection [12, 16]. In addition, 5-HT₇ receptors are coupled to G_α12 (G protein subunit alpha 12) activating the Rho-GEFs-Ras homolog family-Guanine Nucleotide Exchange Factor signaling and Cdc42 (Cell division control protein 42 homolog), which could significantly influence the cytoskeletal architecture [14, 17]. There are reports showing that Cdc42 activation by 5-HT₇ receptor is associated with stimulation of neurites elongation in cortical, striatal and hippocampal primary cell cultures [18, 19]. Additionally, both of G-protein mediated pathways could have an impact on tyrosine receptor kinase B (TrkB) receptor expression, which has been associated with 5-HT₇-mediated neuroprotective effects [20]. Serotonin (5-HT), through activation of 5-HT₇, can evoke anti-inflammatory and anti-apoptotic effects. That effect has been captured as a decrease in caspase–3 and caspase–9 gene expression and was reduced after the addition of 5-HT₇ antagonist, SB269970 [21]. Despite extensive studies, the full biological role of this receptor still remains unclear. The effects of 5-HT₇ action has been studied using few, non-selective tool compounds, e.g.: LP-211, LP-44, LP-12, AS-19, E-55888, RA-7, 5-CT, 8-OH-DPAT [17, 22, 23]. 5-HT₇ has been shown to exert a modulatory role in depression, anxiety, schizophrenia, sleep disruptions and obsessive–compulsive disorder [9, 11, 24]. It has been suggested, that 5-HT₇ activation plays a role in neuroprotection and could increase the viability of neurons as well as have a beneficial role on neurite elongation and synaptogenesis [11, 21, 25,26,27]. Nevertheless, because of low selectivity, low metabolic stability and weak blood–brain barrier permeability, the search for applicable compounds is still ongoing [22, 24]. A recently emerged series of indole-imidazoles, weakly basic 5-HT₇ agonists have been shown to exhibit high affinity and selectivity for 5-HT₇, as well as high water solubility. Chosen compounds, such as AH-494 or AGH-192 exhibited favorable ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) properties [22, 28, 29].

In this study we compared the neuroprotective and neurite outgrowth potential of known (AH-494—compound 15, AGH-238—compound 37) [29], and previously unpublished (AGH-194) 5-HT₇ agonists with 5-carboxyamidotryptamine (5-CT) (Table 1). 5-CT is a non-selective 5-HT₇ agonist, showing high affinity to 5-HT_1A [17, 30, 31]. Despite their tumor origin, human neuroblastoma SH-SY5Y cells are widely used in neurotoxicity and neuroprotection studies as a reliable and cost effective neuronal-like screening platform [32, 33]. Because these cells have dopaminergic phenotype independent of their state of differentiation, they are a common cellular model to study molecular mechanisms and new possible treatments of PD [34, Full size image

The Impact of Differentiation and the Type of Experimental Medium on 5-HT₇ Receptor mRNA Expression in SH-SY5Y Cells

The RA-SH-SY5Y cells incubated in DMEM medium showed a significantly higher level of mRNA expression for the 5-HT₇ receptor than the cells maintained in NB. However, UN-SH-SY5Y showed no significant difference in mRNA expression depending on the type of experimental medium used. Furthermore, in case of cells cultured in DMEM there was significantly higher level of HTR7 expression in RA-SH-SY5Y compared to UN-SH-SY5Y (Fig. 1B). Additionally for UN- and RA-SH-SY5Y incubated in the NB medium the additional statistical analysis was carried out using the t-student test, which showed a significant increase in HTR7 expression in RA-SH-SY5Y cells, when compared UN-SH-SY5Y cells (p < 0.01).

Evaluation of 5-HT₇ Agonists Biosafety Profile in UN-SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

The incubation of UN-SH-SY5Y cells maintained in DMEM medium for 24 as 48 h with 5-CT and AH-494 in wide range of concentrations (0.1–80 µM) did not affect cell viability and cytotoxicity when compared to control groups (Figs. 2A, B, 3A, B). AGH-238 at concentration 20 µM showed an increase in cell viability after 24 h (~ 20%) and 48 h (~ 30%) (Fig. 2C), and in LDH release test this compound at concentration 80 μM after 48 h evoked a significant (~ 30%) increase in cytotoxicity level (Fig. 3C). For AGH-194 we observed a concentration- and time-dependent decrease in cell viability (~ 30–100%) with maximum damage of cells evoked by its higher concentrations (40 and 80 μM) (Fig. 2D). Moreover, for concentrations 20 and 40 µM of AGH-194 there was a greater decrease in cell viability after 48 h compared to 24 h. In the cytotoxicity assay an increase in LDH release was observed for concentrations 40 and 80 µM (~ 2–4- times) after 24 h incubation but also for concentrations 10–80 µM (~ 1.5–3 times) after 48 h. In addition for 20 and 40 µM of AGH-194 there was a significant increase in LDH release after 48 h compared to the level obtained after 24 h. Whereas in the case of 80 µM higher level of released LDH was observed after 24 h than after 48 h (Fig. 3D).

Twenty four hours incubation of UN-SH-SY5Y cells maintained in NB medium with 5-CT, AGH-238 and AH-494 at concentration range 1–20 µM did not affect cell viability (Table 2). However, AGH-194 at concentration of 20 μM evoked a significant decrease of cell viability at (~ 20%) (Table 2). An additional statistical analysis by t-test for AGH-194 at concentration 20 μM between cells maintained in DMEM and NB experimental medium revealed no significant differences between tested groups (p = 0.477).

Table 2 Biosafety profile of 5-HT₇ agonists in UN-SH-SY5Y cells in NB experimental medium

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The Effect of 5-HT₇ Agonists on SH-SY5Y Cell Proliferation

We did not observe any changes in cell proliferation rate measured by MTT reduction test after 24, 48 and 72 h of incubation of SH-SY5Y cells with 5-CT and AH-494 in wide range of concentrations (0.001–80 µM) in DMEM cell culture medium containing 10% FBS (Figs. 4A and B). However incubation with AGH-238 at concentration of 40 µM showed a transient increase in cell viability (~ 25%) after 48 h (Fig. 4C). This effect was significantly different when compared to 24 or 72 h time points. The same compound at concentration of 80 µM after 24 h induced a transient viability decrease (~ 30%), and that effect disappeared after longer incubation times (48 and 72 h) (Fig. 4C). AGH-194 evoked a time- and a concentration-dependent reduction in cell viability (Fig. 4D). Maximal cell damage was observed for concentration 80 µM after 24 h and this effect was similar after 48 h and 72 h. Concentrations 10–40 µM of AGH-194 evoked a concentration-dependent decrease in cell viability (~ 20–60%) after 48 h and these effects were significantly higher to relevant concentrations at 24 h time point. We noticed that AGH-194 at concentration 40 µM induced significantly higher reduction in cell viability (~ 20%) after 72 h when compared to 48 h (Fig. 4D).

Evaluation of Neuroprotective Potential of 5-HT₇ Agonists Against Cell Damage Induced by H₂O₂ in SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

While UN-SH-SY5Y cells were incubated with H₂O₂ (375 μM) in DMEM experimental medium, the damage factor induced about 50% cell viability decrease as well over 3.5-fold increase in LDH release when compared to control group. These effects were significantly decreased by antioxidant NAC (Table S1). In UN-SH-SY5Y cells cultured in NB experimental medium we found that H₂O₂ (150 μM) evoked around 30% decrease in cell viability and about fourfold increase in LDH level which was significantly decreased by NAC (Table S1). Similar observations were done for RA-SH-SY5Y cells cultured in NB experimental medium where H₂O₂ (200 µM) evoked about 35% decrease in cell viability and fourfold increase in LDH level which in the former assay were significantly reversed by NAC (Table S2). The biochemical results for UN- and RA-SH-SY5Y cells incubated with DMEM or NB medium and treated for 24 h with H₂O₂ and NAC was also confirmed at morphological level using DIC light microscopy method. After treatment of cells with H₂O₂ we observed an increased number of rounded cells without protrusions with weaker adhesion to the surface which were prevented by NAC (Fig. S1).

In UN-SH-SY5Y cells incubated in DMEM medium 5-CT at concentrations of 0.01–1 µM did not affect the H₂O₂-induced decrease in cell viability (Fig. 5A), however in cytotoxicity test, concentrations of 0.1 and 1 µM of this compound significantly decreased the level of LDH induced by H₂O₂ (Fig. 5B). AH-494 at concentrations of 0.01–1 µM showed small but statistically significant increase (~ 10%) in cell viability when compared to the H₂O₂-treated cells (Fig. 5C), whereas in cytotoxicity assay we did not observe any protective effects for AH-494 (Fig. 5D). The incubation of cells with AGH-238 as well as AGH-194 at concentrations of 0.01–1 µM did not significantly affect the extent of cell damage evoked by H₂O₂ in both used screening assays (WST-1 and LDH release tests). (Figs. 5E–H).

5-CT at concentrations of 0.01 and 0.1 µM significantly increased (9–11%) UN-SH-SY5Y cell viability cultured in NB medium when compared to H₂O₂ group (Fig. 6A). The same compound at concentrations of 0.01–1 µM decreased level of H₂O₂-stimulated LDH release about 68–110% (Fig. 6B). AH-494 at concentration of 0.01 µM significantly increased (~ 10%) cell viability as well as decreased amount of released LDH (94–57%) in comparison to results from H₂O₂ group (Figs. 6C–D). For compound AGH-238 at concentration of 1 µM an significant increase in cell viability was observed (Fig. 6E) as also decrease in cytotoxicity for concentrations of 0.01–1 µM (64–105%) when compared to the H₂O₂ treated group (Fig. 6F). AGH-194 at the level of cell viability assessment did not affect the extend of cell damage evoked by H₂O₂ (Fig. 6G)_, although in cytotoxicity assay, at concentration of 0.1 µM it partially attenuated the H₂O₂-evoked increase in LDH release (Fig. 6H). Neuroprotective effects of 5-HT₇ agonists found in biochemical assays were confirmed also at morphological level (Fig. 7) where we observed partial improvement of cell morphology after treatment with 0.1 µM 5-CT, AH-494, AGH-238 or AGH-194 in comparison to the H₂O₂ group.

In RA-SH-SY5Y cells cultured in NB medium we did not observe any protective effects of 5-CT, AH-494, AGH-238 and AGH-194 at concentrations of 0.01–1 µM against the cell damage induced by H₂O₂ (200 µM) as confirmed by cell viability and cytotoxicity assays (Table 3). Furthermore, in the LDH release test there was a significant increase of cytotoxicity evoked by AGH-238 at concentration of 1 µM when compared to H₂O₂ group (Table 3).

Table 3 Neuroprotective effects of 5-HT₇ agonists against the H₂O₂-induced cell damage in RA-SH-SY5Y cells cultured in NB experimental medium

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Effects of 5-HT7 Agonists on H₂O₂-Induced Caspase–3 Activity in UN-SH-SY5Y Cells Cultured in NB Experimental Medium

In order to explore some of mechanisms which could be engaged in 5-HT₇-mediated neuroprotection against the H₂O₂-evoked cell damage, we measured activity of the main apoptotic executor protease, caspase-3 [38]. Incubation of UN-SH-SY5Y cells cultured in NB experimental medium for 9 h with H₂O₂ (150 µM) induced approximately fourfold increase in caspase–3 activity which was fully inhibited by caspase–3 inhibitor, Ac-DEVD-CHO (20 µM) but not by any of the tested 5-HT₇ agonists (Fig. 8).

Evaluation of 5-HT₇ Agonists Neuroprotective Potential in Other Cells Damage Models in UN- and RA-SH-SY5Y Cultured in NB Experimental Medium

In order to verify a neuroprotective action of 5-HT₇ agonist in the PD cellular model we incubated UN-SH-SY5Y cells with 6-OHDA (75 µM) in NB medium which induced approximately 55% decrease of viability in comparison to control group, and this effect was significantly attenuated by NAC (Table S2). In case of RA-SH-SY5Y cells after 24 h of treatment with 6-OHDA (150 µM) 35% decrease of cell viability was observed and this effect was also significantly reversed by NAC (Table S2).

Twenty four hours of treatment of UN-SH-SY5Y cells cultured in NB medium with 5-CT, AH-494, AGH-238 and AGH-194 at concentrations of 0.01–1 µM and neurotoxin 6-OHDA did not change the cell viability (Figs. 9A, C, E, G) and cytotoxicity (Figs. 9B, D, F, H). Similar results we observed in RA-SH-SY5Y cells in the same experimental medium (NB) where 5-HT₇ agonists did not show any protection against the 6-OHDA-induced cell damage (Table S3).

Forty eight hours of treatment of UN-SH-SY5Y cells with MPP + (1 mM) evoked about 50% reduction in cell viability which was not changed by 5-HT₇ agonists at any tested concentration (0.01–1 µM) (Table S4). Similar results we found in RA-SH-SY5Y cells where 5-HT₇ agonist did not show any protection against the MPP +−induced cell damage (Table S4).

Twenty four hours treatment of UN- and RA-SH-SY5Y cells in NB experimental medium with Dox (1 and 2 µM for UN- and RA-SH-SY5Y, respectively) evoked approximately 40–50% decrease in cell viability as measured by WST-1 test (Table S5). 5-CT, AH-494, AGH-238 and AGH-194 in none of the concentrations used (0.01–1 µM) had a significant effect on UN- and RA-SH-SY5Y cells viability decrease evoked by Dox (Table S5).

The Effects of 5-HT₇ Agonists on Neurite Outgrowth in SH-SY5Y Cells

To examine the impact of tested 5-HT₇ agonists on neurite outgrowth, the SH-SY5Y cells were treated for 24 and 48 h with 5-CT, AH-494, AGH-238 and AGH-194 at concentrations of 0.01–1 µM. 5-CT and AGH-238 at concentrations of 0.01–1 µM did not significantly affect neurite outgrowth in SH-SY5Y cells when compared to control group at any of tested time points. AH-494 and AGH-194 at concentrations of 0.01 and 0.1 µM after 24 h of incubation significantly increased the neurite length to the level which was achieved by positive control RA (10 µM). After 48 h of treatment we still observed an increased neurite length by RA, but we did not find significant effects for any of tested 5-HT₇ agonists. However, some tendency in increase of this parameter could be noted for AH-494 (0.01 µM) and AGH-194 (0.1 and 1 µM) (Fig. 10).

Discussion

According to the Ehrlich’s concept of a ‘magic bullet’, there’s a demand to find selective chemical compounds, which could induce specific actions within cell signaling via specific receptors. These compounds may be used as research tools or therapeutics. In contrast, commonly used protein ligands often act non-specifically and could cause undesirable, negative effects on the organism as a whole [46,47,48,49]. It is often relatively easy to find selective antagonist of a protein of interest, compared to a selective agonist. 5-HT₇ receptor emerged as a putative molecular target for neurological disorders such as anxiety, depression, neurodegenerative disorders and pain [9, 23, 24]. Although pyrroloimidazoles have been reported in the context of neurite outgrowth stimulation, they have not been linked to, and it is highly doubtful, that their effects could be mediated by 5-HT₇ signalling [50]. It should be underlined that currently there have been only a few experimental studies which investigated the neuroprotective potential of the 5-HT₇ receptor [21, 27, 51, 52]. For that reason obtaining any new data regarding 5-HT₇ contribution in attenuating nervous system disorders could be profitable. So far for in vitro and in vivo research dedicated to 5-HT₇ mechanism of action, SB269970—specific antagonist/inverse agonist of 5-HT₇ [53] and commercially available agonist 5-CT were tested for examining the pharmacological activation of the receptor [54,55,56]. In case of 5-CT there are some issues that should be taken into consideration. 5-CT was shown to be non-selective, despite the high affinity for 5-HT₇ [14, 17] and the results thus obtained by use of this substance could be misinterpreted in relation to its mechanism of action. Throughout the years, many research groups synthesized and examined compounds acting on 5-HT₇ as they were trying to find agonists which have better pharmacokinetic parameters and also high selectivity to 5-HT₇ [17, 57,58,59]. So far no compounds acting through 5-HT₇ were found and considered to be used in clinic [24, 31, 60, 61]. Thus we tested in the context of neuroprotective and neurite outgrowth properties the three new, low basicity 5-HT₇ agonists: AH-494, AGH-238 and AGH-194, that have a high affinity to 5-HT₇ and good selectivity over 5-HT_1A. First, we measured the expression of 5-HT₇ in our model, the human neuroblastoma SH-SY5Y cells. We showed the impact of cell differentiation state and the type of the experimental medium on the expression of transcript for 5-HT₇. We confirmed previous results showing higher expression of HTR7 in differentiated SH-SY5Y when compared to undifferentiated ones [25] and showed for the first time the impact of the type of used experimental medium on this target. We observed a higher level of 5-HT₇ transcript in cells incubated in neuroblastoma dedicated medium (DMEM with low serum content) in comparison to cells placed in neuronal medium (NB). It cannot be excluded that stimulation of the expression might be a cell endogenous response for trophic factors deprivation, associated with the serum reduction from 10 to 1%, whereas in NB medium trophic factors are provided by B27 supplement [62].

The second step was undertaken to establish the biosafety profile of the tested 5-HT₇ agonists. In this context, the highest level of cytotoxicity as well as the effect on cell viability decrease was found for AGH-194. It was shown that this compound evoked cell death in UN-SH-SY5Y cells incubated both in DMEM and NB at concentrations above 10 µM. A decrease in cell viability was also observed for AGH-194 at concentrations of 10 µM and 20 µM in RA-SH-SY5Y, which confirmed the high cytotoxicity of this compound. It should be noted that after differentiation, SH-SY5Y cells usually show higher resistance to various cell damaging factors [38, 43] which was not the case with AGH-194. Biosafety profile of the other tested 5-HT₇ agonists (AH-494, AGH-238 and 5-CT) up to 80 µM was found to be favorable for UN- and RA-SH-SY5Y cells cultured in DMEM experimental medium. Previous data for AH-494 (0.1–100 µM) in HEK-293 and SH-SY5Y showed no cytotoxic effect of this compound [22], which is in line with our findings. However, in HepG2 cells concentration of 100 µM of AH-494 evoked significant reduction in cell viability, pointing that possible detrimental effects could be the cell type-dependent [22]. It is worth to mention that the experiments done by Latacz et al. [22] were performed under typical cell culturing conditions for proliferating cells (DMEM + 10% FBS), which could make cells more resistant to potentially harmful compounds. In contrast, our results were obtained in conditions of reduced serum content (DMEM + 1% FBS) which could make cells more vulnerable to potential toxicity of the tested agents. In our study, AGH-238 induced transient increase of cell viability at concentration of 20 µM in UN-SH-SY5Y cells cultured in DMEM experimental medium, which probably is connected with the impact of this compound on cell proliferation, since our cellular model is of tumor origin. For that reason we also checked the impact of 5-HT₇ agonists on SH-SY5Y cells proliferation under standard cell culturing conditions (DMEM + 10% FBS). We observed that 5-CT as well as AH-494 up to 80 µM did not affect the cell proliferation (measured indirectly by MTT test). For AGH-238 at concentration of 40 µM a transient increase in cell viability/proliferation was observed at 48 h of incubation which is partially consistent with the effect observed in results of biosafety profile for this compound, whereas at concentration of 80 µM after 24 h this compound evoked transient viability decrease, which disappeared during longer incubation time (48 h and 72 h). This effect could explained as restarting the proliferation process by cells which survived the initial exposure to the compound and/or by compound deactivation after longer time of incubation. To confirm the aforementioned hypothesis, further experiments would be necessary, where the proliferation rate would be measured with more specific markers like BrdU or Ki67 [63]. The highest decrease of cell viability that was time and concentration dependent, and interpreted as a decrease of proliferation was obtained for AGH-194, where at 80 µM, almost total reduction of cell viability for each of the tested time points was observed. For the lower concentrations (10–40 µM) there was a significant decrease of cell viability (~ 20–60%) after 48 h and 72 h in comparison to the 24 h treatment. The observed changes at the higher concentrations were probably caused by cellular death rather than proliferation suppression. When testing biosafety prolife of 5-HT₇ agonists in UN- and RA-SH-SY5Y cell cultured in NB medium at concentration range up to 20 µM for 24 h we noticed a significant reduction of cell viability by AGH-194 (at concentration 20 and 10–20 µM for UN- and RA-SH-SY5Y cells, respectively) but not by the other tested compounds. The above findings prove, that AGH-194 shows greater cytotoxicity than other the tested 5-HT₇ ligands under various cell culture conditions at least in human SH-SY5Y cells, which could prohibit its potential future use in the clinic. Those effects could potentially be attributed to the increased lipophilicity of AGH-194 versus AGH-238 or AH-494.

Since we observed the impact of the cell differentiation state as well the type of experimental medium on 5-HT₇ expression, thus we examined the potential neuroprotective effects of 5-HT₇ compounds in UN- and RA-SH-SY5Y cells cultured in DMEM and NB experimental medium. For this purpose we used an inducer of oxidative stress, H₂O₂, as well as neurotoxins that mimic PD, like 6-OHDA and MPP + as well as a pro-apoptotic factor, Dox. By their use we could model cellular damage which mimics different pathological mechanisms of PD like apoptosis, necrosis, autophagy, oxytosis, pyroptosis, necroptosis [7, 64, 65]. H₂O₂ is broadly used in cellular models as an oxidative stress inducer for testing a protective potential of medicinal substances [66]. In that type of cell damage we were able to demonstrate for the first time neuroprotection mediated by 5-HT₇ agonists and that effect was dependent on the cell differentiation state as well on the type of experimental medium. We observed some protection for 5-CT (0.1–1 µM) in LDH release test and for AH-494 (0.01–1 µM) in cell viability test against the H₂O₂-evoked detrimental changes in UN-SH-SY5Y cells cultured in DMEM experimental medium. Since these results were not confirmed in both biochemical tests thus this neuroprotective effect could be interpreted as biologically negligible. However, in the NB medium we found a significant protection by 5-CT, AH-494 and AGH-238 in both cell viability and cytotoxicity assays. For AGH-194 we observed only a decrease of the H₂O₂-evoked LDH release but no effects on the cell viability which suggests lower neuroprotective potential of this compound. It is worth to highlight that all of the observed changes after 5-HT₇ treatment were partial and none of tested compound completely reduced detrimental effects induced by H₂O₂ to the level mediated by antioxidant, NAC. Referring to these neuroprotective effects mediated by 5-HT₇ agonists, it can be concluded that LDH test is more sensitive for detection of neuroprotective response of 5-HT₇ ligands which were also confirmed by our cell imaging studies. Interestingly, in RA-SH-SY5Y we did not find any protection by 5-HT₇ agonists under both types of experimental medium which suggest that mechanisms of protection mediated by this compounds could be mimicked by RA during the process of cell differentiation where activation of prosurvival pathways like PI3K/AKT and ERK 1/2 takes place [37]. Similar observation was done by us for ligands of metabotropic glutamate receptors group II or III which were protective against staurosporine, Dox or MPP + -induced cell damage in UN- but not in RA-SH-SY5Y [38, 43]. It should be noted that the protective effects observed after 5-HT₇ agonists treatment against the H₂O₂-evoked cell damage were not positively correlated with the level of expression of 5-HT₇ mRNA in particular cell types. Tested compounds had stronger protective properties in UN-SH-SY5Y cells placed in NB medium than DMEM, while no protection was observed in RA-SH-SY5Y under any experimental condition, when HTR7 expression was the highest in RA-SH-SY5Y cells cultured in DMEM medium. It can refer to the fact that the obtained mRNA level for the transcript could not be translated to significant protein levels in cells [66], but this assumption needs to be experimentally verified. An additive factor that can contribute to divergent effects could be non-specific action of ligands, especially 5-CT which can activate either 5-HT₇ and also 5-HT_1A receptor [58]. What is more, these receptors can dimerize with each other creating complexes, what can additively cause problems with interpretation of effects [14]. Nevertheless, there were some research outcomes which indicated protective properties of commercially available 5-HT₇ ligands [9, 17, 20, 24, 67], thus our results are supporting already existing literature data. Regarding previous reports on a neuroprotective potential of 5-HT₇ agonists, Vasefi et al. [27] showed that 24 h incubation of primary hippocampal cells with LP12 (300 nM) prevented neurotoxicity caused by NMDA activation and this effect was dependent on PDGFβ (Platelet-derived growth factor β) receptor kinase activation. Yuksel et al. [21] examined the protective action of LP-44 (10⁻¹⁰, 10⁻⁹, 10⁻⁸ mM) in an oxidative stress model induced by a high concentration of glutamate (80 mM) in SH-SY5Y cells. This group also observed an increased expression of 5-HT₇ receptor after glutamate exposure. These results can suggest that the receptor expression could increase in unfavorable cell conditions, which is consistent with our observations, where under serum deprivation (medium DMEM 1%) mRNA level for 5-HT₇ was significantly increased compared to the NB medium. Moreover, on the basis of the above findings it can be noticed that the neuroprotective effect was induced by low concentrations of 5-HT₇ agonists which is in line with our data. In the future studies, the specificity of 5-HT₇ activation of observed protective effects should be taken into consideration as well as intracellular pathways which could be responsible for these effects. Regarding the latter, in our study we showed that tested 5-HT₇ agonists did not decrease the level of caspase–3 activity induced by H₂O₂. This is in contrast to the findings of Yuksel et al. [21], who showed that LP-44-mediated neuroprotection against glutamate-evoked oxytosis in SH-SY5Y cells was associated with caspase-3 and caspase-9 inhibition. It should be underlined that protective effects without simultaneous decrease of caspase-3 activity were observed in our previous study when we tested the effectiveness of agonists of metabotropic glutamatergic receptors group II and III against cell damage evoked by proapoptotic factors (staurosporine and Dox) [43]. Since in these models we observed the participation of caspase- 3-independent mechanism coupled with inhibition of AIF (apoptosis inducing factor) translocation to nucleus [43], it is not excluded that a similar mechanism is involved in the case of 5-HT₇ agonist action against the H₂O₂ evoked cell damage. It was shown in SH-SY5Y and neuronal cells that after H₂O₂ exposure there is an induction of AIF translocation [43, 68]. Moreover, the inhibition of calcium dependent proteases, calpains or lysosomal enzyme, cathepsin D [69] or the inhibition of necroprotsis [44] could be the other possible mechanisms responsible for 5-HT₇ mediated protection against H₂O₂.

The neuroprotective action of 5-HT₇ agonists observed in this study seems to be limited to the model of oxidative stress induced by H₂O₂, since we did not find any attenuation by 5-CT, AH-494, AGH-238 or AGH-194 of the cell damage evoked by 6-OHDA, MPP + and Dox both in UN- and RA-SH-SY5Y cells, cultured in NB experimental medium. It suggests that the neuroprotective effects of 5-HT₇ agonists are strongly dependent on the type of cell damage and they can involve the protection mechanisms that are specific to the cascade of events leading to cellular death induced by H₂O_2. It should be underlined that the previous findings also pointed that effects mediated by 5-HT₇ receptor in the CNS may be evoked by either agonists and antagonists (or inverse agonists) in dependence on the type of cell damage or the used experimental model [70]. For example SB-269970 (5-HT₇ selective antagonist) increased the production of dopaminergic cells in rat’s midbrain neutrospheres whereas activation of 5-HT₇ receptor by 8-OH-DPAT inhibited this process [71]. While neuronal damage was modeled by 6-OHDA in rat’s prefrontal cortex, 5-HT₇ agonist AS-19 showed antidepressant effects which were reversed by SB-269970 [72]. What is interesting, while 6-OHDA evoked lesions in lateral habenula, this effect was reversed by SB-269970 which showed also antidepressant properties, whereas AS-19 induced prodepressive behavior in rats [73]. Moreover it is believed that antagonists of 5-HT₇ are promising for the treatment of schizophrenia or epilepsy [9]. SB-269970 is commonly used to block the protective effects of 5-HT₇ agonists [21] and in the future it is advisable to check if this compound inhibits the effects of the herein tested agonists. To this end we did not find any protection mediated by SB-269970 (0.1–10 µM) against cell damage induced by H₂O₂ and 6-OHDA in UN- and RA-SH-SY5Y cells cultured in NB experimental medium (data not shown).

At the final stage of the study, we investigated if the tested 5-HT₇ ligands have an impact on neurite outgrowth in SH-SY5Y cells. Two of the tested compounds (AGH-194 and AH-494) demonstrated beneficial properties in that aspect and their action was stronger compared to the commercially available agonist 5-CT. These effects were observed only after 24 h incubation with the agonists, which suggests that their action was transient. As a positive control we used RA (10 μM), which significantly increased the neurite length in each investigated time point (24 h and 48 h). The effect of 5-HT₇ stimulation on neurite elongation was previously examined and confirmed by Chang et al. [25], who incubated SH-SY5Y cells with 5-HT (1 μM) and LP211 (0.1 and 1 μM), and the effect of these compounds was inhibited by SB-269970. But what differs our work from their is the analysis, because they calculated only the percentage of neurons with a length of neurites greater than 50 μm. In comparison to that we analyzed elongation for specific time points. Moreover, it is possible that this effect we observed only in the case of two compounds, is 5-HT₇ dependent, because 5-CT which has a similar affinity to 5-HT₇ and 5-HT_1A did not demonstrate any action promoting neurite outgrowth in our experimental setting. Since the previous studies demonstrated that changes in the cytoskeleton are dependent on G_α12 and Cdc42 activation after 5-CT (100 nM) stimulation in primary hippocampal cell cultures [74], their possible involvement in the observed beneficial effects of 5-HT₇ agonists in SH-SY5Y cells should be further investigated.

Conclusions

Our data showed that SH-SY5Y cells are a suitable cellular model for testing the biological activity of 5-HT₇ agonists, because they express 5-HT₇ receptors, which is cell phenotype and experimental medium composition dependent. Furthermore, we showed that the new low-basicity 5-HT₇ agonists (AH-494, AGH-238 and AGH-194) as well as the commercially available 5-CT show some neuroprotective effects against H₂O₂-evoked cell damage in SH-SY5Y cells and this effect is also dependent on the cell phenotype and experimental medium composition. The observed neuroprotective effects of 5-HT₇ agonists were not associated with the inhibition of caspase–3 activity. Among the tested compounds, AH-494 appeared to have the greatest protective potential, whereas AGH-194 the lowest one. None of the examined compounds showed neuroprotective properties in cellular damage models evoked by 6-OHDA, MPP + and Dox in SH-SY5Y cells. Finally, the two of the tested 5-HT₇ agonists (AH-494 and AGH-194) promoted neurite outgrowth. The results obtained in SH-SY5Y cells provide new insights in the context of neuroprotective and neurite outgrowth potential of the new 5-HT₇ agonists, which should be verified in the future in animal studies.

Data Availability

The raw data that support the findings of this study are available upon reasonable request from the corresponding author.

Abbreviations

6-OHDA:: 6-Hydroxydopamine
DIC:: Differential interference contrast
Dox:: Doxorubicin
H₂O₂ :: Hydrogen peroxide
LDH:: Lactate dehydrogenase
MPP + :: 1-Methyl-4-phenylpyridine
MTT:: 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
ND:: Neurodegenerative diseases
PD:: Parkinson’s disease
RA:: Retinoic acid
RA-SH-SY5Y:: Retinoic acid-differentiated SH-SY5Y cells
ROS:: Reactive oxygen species
UN-SH-SY5Y:: Undifferentiated SH-SY5Y cells

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Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Barbara Korzeniak.

Funding

The study was financed by the statutory funds of Maj Institute of Pharmacology of the Polish Academy of Sciences.

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Authors and Affiliations

Department of Experimental Neuroendocrinology, Maj Institute of Pharmacology of the Polish Academy of Sciences, Krakow, Poland
Klaudia Jakubowska & Danuta Jantas
Department of Medicinal Chemistry, Maj Institute of Pharmacology of the Polish Academy of Sciences, Krakow, Poland
Adam S. Hogendorf
Department of Molecular Neuropharmacology, Maj Institute of Pharmacology of the Polish Academy of Sciences, Krakow, Poland
Sławomir Gołda

Authors

Klaudia Jakubowska
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Adam S. Hogendorf
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Sławomir Gołda
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Danuta Jantas
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Contributions

Danuta Jantas contributed to the study conception and design. Material preparation, data collection and analysis were performed by Klaudia Jakubowska, Adam S. Hogendorf, Sławomir Gołda and Danuta Jantas. The first draft of the manuscript was written by Klaudia Jakubowska and Danuta Jantas and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Danuta Jantas.

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Jakubowska, K., Hogendorf, A.S., Gołda, S. et al. Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT₇ Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells. Neurochem Res 49, 2179–2196 (2024). https://doi.org/10.1007/s11064-024-04159-z

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Received: 16 March 2024
Revised: 16 March 2024
Accepted: 20 May 2024
Published: 04 June 2024
Issue Date: August 2024
DOI: https://doi.org/10.1007/s11064-024-04159-z

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Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT₇ Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

Abstract

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Selective Aryl Hydrocarbon Receptor Modulator 3,3′-Diindolylmethane Impairs AhR and ARNT Signaling and Protects Mouse Neuronal Cells Against Hypoxia

Introduction

The Impact of Differentiation and the Type of Experimental Medium on 5-HT₇ Receptor mRNA Expression in SH-SY5Y Cells

Evaluation of 5-HT₇ Agonists Biosafety Profile in UN-SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

The Effect of 5-HT₇ Agonists on SH-SY5Y Cell Proliferation

Evaluation of Neuroprotective Potential of 5-HT₇ Agonists Against Cell Damage Induced by H₂O₂ in SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

Effects of 5-HT7 Agonists on H₂O₂-Induced Caspase–3 Activity in UN-SH-SY5Y Cells Cultured in NB Experimental Medium

Evaluation of 5-HT₇ Agonists Neuroprotective Potential in Other Cells Damage Models in UN- and RA-SH-SY5Y Cultured in NB Experimental Medium

The Effects of 5-HT₇ Agonists on Neurite Outgrowth in SH-SY5Y Cells

Discussion

Conclusions

Data Availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing Interests

Additional information

Publisher's Note

Supplementary Information

Supplementary file1 (DOCX 1128 KB)

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About this article

Cite this article

Keywords

Navigation

Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT7 Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

Abstract

Similar content being viewed by others

Cytotoxic Effects of 3,4-Catechol-PV (One Major MDPV Metabolite) on Human Dopaminergic SH-SY5Y Cells

Neurotoxicity Assessment of 1-[(2,3-Dihydro-1-Benzofuran-2-yl)Methyl]Piperazine (LINS01 Series) Derivatives and their Protective Effect on Cocaine-Induced Neurotoxicity Model in SH-SY5Y Cell Culture

Selective Aryl Hydrocarbon Receptor Modulator 3,3′-Diindolylmethane Impairs AhR and ARNT Signaling and Protects Mouse Neuronal Cells Against Hypoxia

Introduction

The Impact of Differentiation and the Type of Experimental Medium on 5-HT7 Receptor mRNA Expression in SH-SY5Y Cells

Evaluation of 5-HT7 Agonists Biosafety Profile in UN-SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

The Effect of 5-HT7 Agonists on SH-SY5Y Cell Proliferation

Evaluation of Neuroprotective Potential of 5-HT7 Agonists Against Cell Damage Induced by H2O2 in SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

Effects of 5-HT7 Agonists on H2O2-Induced Caspase–3 Activity in UN-SH-SY5Y Cells Cultured in NB Experimental Medium

Evaluation of 5-HT7 Agonists Neuroprotective Potential in Other Cells Damage Models in UN- and RA-SH-SY5Y Cultured in NB Experimental Medium

The Effects of 5-HT7 Agonists on Neurite Outgrowth in SH-SY5Y Cells

Discussion

Conclusions

Data Availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing Interests

Additional information

Publisher's Note

Supplementary Information

Supplementary file1 (DOCX 1128 KB)

Rights and permissions

About this article

Cite this article

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Navigation

Neuroprotective and Neurite Outgrowth Stimulating Effects of New Low-Basicity 5-HT₇ Receptor Agonists: In Vitro Study in Human Neuroblastoma SH-SY5Y Cells

The Impact of Differentiation and the Type of Experimental Medium on 5-HT₇ Receptor mRNA Expression in SH-SY5Y Cells

Evaluation of 5-HT₇ Agonists Biosafety Profile in UN-SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

The Effect of 5-HT₇ Agonists on SH-SY5Y Cell Proliferation

Evaluation of Neuroprotective Potential of 5-HT₇ Agonists Against Cell Damage Induced by H₂O₂ in SH-SY5Y Cells Cultured in DMEM and NB Experimental Medium

Effects of 5-HT7 Agonists on H₂O₂-Induced Caspase–3 Activity in UN-SH-SY5Y Cells Cultured in NB Experimental Medium

Evaluation of 5-HT₇ Agonists Neuroprotective Potential in Other Cells Damage Models in UN- and RA-SH-SY5Y Cultured in NB Experimental Medium

The Effects of 5-HT₇ Agonists on Neurite Outgrowth in SH-SY5Y Cells