In this study, we have elucidated the association between the differentiation of SH-SY5Y cells and alterations in the expression of ATP-dependent potassium channels Kir 6.2, shifts in mitochondrial morphology and functions, and variations in cellular metabolism. Furthermore, we have documented the modified responses of both non-differentiated and differentiated SH-SY5Y cells treated with rotenone to the modulators of the functions of ATP-dependent potassium channels. Despite its neuroblastoma origin, the SH-SY5Y cell line has been extensively utilized as a reliable experimental model to the study of Parkinson’s-like neurodegeneration (Ioghen et al. 2023). The impact of ATP-dependent potassium channels (KATP) on neuronal function has been a subject of interest for over three decades, particularly in neurodegenerative disorders such as Parkinson’s disease, ischemic insults, and Alzheimer’s disease (Szeto et al. 2018; Lv et al. 2022). The outcomes of our current study provide valuable insights into the distinctions between dopaminergic progenitor cells and adult dopaminergic neurons derived from the SH-SY5Y cell line, especially in their responses to KATP channel modulators. Given the pivotal role of mitochondrial functions in the mechanism of neurodegeneration, our set of experiments specifically focused on potential alterations in mitochondrial morphology, energy metabolism, metabolic activity, and mitochondrial respiration. The primary objective was to elucidate the characteristic features of both types of the SH-SY5Y cells under normal conditions and during pathological conditions induced by a 24-hour treatment with 50 nM rotenone.

The presented results highlight fundamental differences in mitochondrial network complexity between differentiated and non-differentiated SH-SY5Y cells. Specifically, differentiated cells manifest a significantly more branched mitochondrial network, as indicated by the elevated ratio of junction count per overall mitochondrial branches (Fig. 3a). Furthermore, the mitochondrial network in differentiated cells exhibits reduced fragmentation, as evidenced by a diminished proportion of solitary mitochondrial structures in relation to the overall mitochondrial branches (Fig. 3b). Another distinguishing feature is represented by the enhanced length of individual mitochondrial branches and rod-like structures in differentiated SH-SY5Y cells in comparison to their non-differentiated counterparts (Fig. 3d). In differentiated cells, the mitochondrial network is notably enlarged, accompanied by a substantial increase in the overall mitochondrial length per cell (Fig. 3e). Our findings additionally indicate a increased respiratory capacity in differentiated cells, as evidenced also by a higher rate of mitochondrial oxygen consumption observed through respirometry (Fig. 3f). Notably, our findings concerning mitochondrial length align with a study conducted on mature neurons in mice. In this investigation, damaged mitochondria under ischemic conditions were linked to increased fragmentation and diminished mitochondrial length (Kim et al. 2022). Furthermore, our results are consistent with another study demonstrating that the induction of retinoic acid-mediated differentiation in SH-SY5Y cells resulted in an augmentation of ATP production (Forster et al. 2016).

We characterized the differences in protein expression of the KCNJ11 gene, responsible for encoding Kir 6.2, across the examined cell types. Western blot analysis revealed a nearly exclusive Kir 6.2 expression in non-differentiated SH-SY5Y cells, as depicted in Fig. 3c. The predominant Kir 6.2 expression in non-differentiated cells is notably unexpected, considering the conventional expectation of elevated levels in neuronal structures and other excitable cell types for this type of potassium rectifier (Huang et al. 2007; Fagerberg et al. 2014). Our findings offer pioneering insights, as a lack of relevant assessments regarding Kir6.2 expression changes during differentiation protocols exists in the current literature. Nonetheless, investigations focusing on the rat entorhinal cortex have identified increased expression of this channel subunit in immature neurons during ontogenesis (Lemak et al. 2014).

Subsequent to the characterization of mitochondrial networks and the assessment of selected protein levels, we delved into the effects of KATP modulation using both its agonist (diazoxide) and antagonist (glibenclamide). Remarkably, basal calcium homeostasis exhibited a significant alteration solely in non-differentiated cells, with the most notable distinction observed between the effects of glibenclamide and diazoxide. In comparison to control and diazoxide treated non-differentiated cells, glibenclamide treatment resulted in a noteworthy increase of calcium concentration in control cells as well as in rotenone treated cells, as illustrated in Fig. 4a. These findings align with existing literature, wherein glibenclamide is linked to calcium overload, while diazoxide is acknowledged for its capacity to decrease intracellular calcium levels (Mariot et al. 1998; Sola et al. 2015).

Furthermore, calcium imaging experiments were conducted, revealing no significant effects of 24-hour treatment with KATP modulators or rotenone in differentiated SH-SY5Y cells. Significant alterations in calcium elevation following excitation by potassium chloride were observed after glibenclamide treatment, especially in control conditions in non-differentiated cells, as depicted in Fig. 4b. However, the significance of glibenclamide’s impact was lowered in pathological conditions induced by rotenone presence. Except that, these data indirectly support the notion of increased protein expression of the KCNJ11 gene in non-differentiated SH-SY5Y cells. Notably, the impact of 24-hour glibenclamide treatment on calcium response to KCl in non-differentiated cells represents a novel finding. In the context of the beneficial effects of glibenclamide on motor symptoms during the progression of Parkinson’s disease (Abdelkader et al. 2020), literature search yielded no relevant result on possible change in, for example, dopamine secretory capacity after glibenclamide treatment. Our results, showing elevated calcium response after glibenclamide treatment, suggest that exploring such possibility could be worthwhile in future experiments. Prior investigations have documented the beneficial effects of glibenclamide in various neurodegeneration models, particularly in terms of its ultimate effects on neurons. Several studies have reported an anti-inflammatory effect of glibenclamide in connection with Parkinson’s disease (Abdelkader et al. 2020; Qiu et al. 2021; Ferdowsi et al. 2022). All documented anti-inflammatory effects of glibenclamide are rooted in animal models, highlighting its ultimate impact on neurons. Our results suggest that in vitro prepared adult dopaminergic neurons, characterized by low expression of this type of ion channel, may not be directly influenced by the drug. However, the expression profile of dopaminergic neurons, as well as their supportive neural cells in vivo could be characterized by different features. Additionally, systemic administration of glibenclamide is reported to be ineffective in reaching the cerebrospinal fluid in concentrations necessary for efficacy, necessitating further exploration of mediated effects (Lahmann et al. 2015).

The principal objective of our study was to elucidate the impact of KATP modulation on mitochondrial physiology in two forms of SH-SY5Y cells under both normal and pathological conditions. Subsequent experiments were designed to evaluate the influence of KATP modulation on cell metabolism, with a specific emphasis on mitochondrial metabolism. Analysis of energy metabolism intermediates unveiled a significant increase in lactate production following the inhibition of complex I, concomitant with reduced concentrations of pyruvate and glucose in the growth medium (Fig. 5a, d). Respirometry experiments consistently affirmed the inhibitory effect of rotenone on mitochondrial respiration, evident in a significant decrease in basal oxygen consumption across both studied cell forms after rotenone treatment (Fig. 6b, d). All aforementioned effects align with standard outcomes anticipated following the inhibition of mitochondrial activity in general.

The effects of KATP modulators were predominantly observed in non-differentiated cells, where we documented a significant stimulatory effect of glibenclamide on lactate production, coupled with an increased uptake of pyruvate from the medium (see Fig. 5a). The nature of this phenomenon could be partially explained by the previously observed inhibitory effects of glibenclamide on mitochondrial activity. Data from several studies suggest that the effects of glibenclamide on mitochondrial metabolism are positively correlated with those of metformin (Salani et al. 2017). Besides mitochondrial parameters, the overall metabolic activity, as assessed through MTT tests, unveiled only a marginal toxic effect of diazoxide (diazoxide p = 0.07) in non-differentiated cells, but not in differentiated SH-SY5Y cells (Fig. 5b). On the other hand, glibenclamide effect was characterized as a beneficial for the cell count in non-differentiated SH-SY5Y (Fig. 5c). On the basis of previous studies, it is evident that the effects of both KATP modulators strongly depend on the chosen experimental model. The impact of both KATP modulators on cell viability was documented by Yilmaz et al. (2015), where diazoxide treatment at a concentration of 10 μM was identified as beneficial for renal tubular cells (Yilmaz et al. 2015). In the same study, glibenclamide exerted toxic effects on the renal cells at the same concentration and time of treatment. The neuroblastoma cells used in our experiments evidently exhibit different sensitivity to the studied KATP modulators. A study published by Du et al. (2016) documented the protective effect of glibenclamide against harmful ferrous influx into the SK-N-SH cells, which constitute the parental cell line for the SH-SY5Y subline (Du et al. 2016). Diazoxide was identified as a negative agent in the chosen experimental procedure. Our experiments with differentiated SH-SY5Y cells further revealed only the toxic effect of rotenone. No significant effects of diazoxide or glibenclamide on the cell viability were observed in differentiated cells (Fig. 5e, f). This phenomenon could be attributed to the lower expression of Kir 6.2 in differentiated cells, as mentioned earlier.

Significant effects of glibenclamide and diazoxide were evident in alterations of mitochondrial potential and respiration in non-differentiated cells, with both KATP modulators displaying a stimulatory effect following 24-hour exposure. In the case of differentiated SH-SY5Y cells, only glibenclamide exhibited a significant stimulatory effect on mitochondrial potential after the 24-hour treatment (Fig. 6c). The observed effect of diazoxide (Fig. 6a) is partially consistent with previous studies, as the opening of mitochondrial KATP channels has stimulating effects on mitochondrial respiration, as evidenced by oxygen consumption (Akopova et al. 2020).

The situation with glibenclamide is intriguing, as earlier investigations have reported inhibitory effects on mitochondrial respiration upon the blockade of mitochondrial KATP channels (Fernandes et al. 2004; Engbersen et al. 2005; Salani et al. 2017). However, other studies have documented stimulatory effects of KATP blockers on mitochondrial respiration under different experimental conditions (Skalska et al. 2005). Notably, our experiments differed from previous studies in terms of duration, as most cell-based experiments have primarily focused on immediate or short-term effects of KATP modulators on mitochondrial respiration. In contrast, our experiments yielded results after a 24-hour treatment with both KATP modulators of both forms of SH-SY5Y cells. This prolonged exposure more closely resembles clinical conditions of long-term treatment, which can be interpreted as a chronic alteration of KATP channel function.

The observed stimulatory effect of rotenone treatment on mitochondrial potential in differentiated cells (Fig. 6c) represents an unusual finding in our experiments. It is essential to highlight that the method employed for measuring mitochondrial potential is based on mitotracker Red FM fluorescence quantification. Rotenone is acknowledged as a potent stimulator of reactive oxygen species (ROS) production, while mitotracker Red FM is a probe known for its high affinity for ROS (Buckman et al. 2001). Considering the noted decrease in mitochondrial respiration in both types of SH-SY5Y cells, this phenomenon may be linked to an increased ROS generation induced by rotenone in differentiated cells.

The comprehensive analysis of morphological alterations in mitochondrial networks under diverse experimental conditions constitutes a significant contribution of our study. The implication of KATP channels in mitochondrial dynamics is a well-recognized phenomenon, and their role in reshaping the mitochondrial network has been previously elucidated by Peng et al. (2018) in a cellular model based on rat adrenal PC12 cells subjected to Parkinson’s-like neurodegeneration. In their investigation, the authors observed negative effects of the combination of an agonist with rotenone on mitochondrial footprint in PC12 cells, along with an increased fragmentation pattern in the mitochondrial network (Peng et al. 2018). These results were positively correlated with behavioral tests in a parallel animal study, and the effect of the selective mitochondrial KATP blocker 5-hydroxydecanoate was found to be slightly beneficial.

Our findings highlight critical differences between the chosen cell types, as evidenced by the protein expression of KCNJ11 between differentiated and non-differentiated SH-SY5Y cells. Non-differentiated SH-SY5Y cells, characterized by higher KCNJ11 expression, were observed to be more sensitive to the KATP modulators used in our study. The effect of both opening and blocking KATP channels was evidently beneficial for the average branch length of mitochondria in non-differentiated cells (Fig. 7b).

Rotenone treatment had a statistically significant downregulating effect on mitochondrial length in neuroblastoma progenitor cells (Fig. 7b). Focusing on differentiated cells, mitochondrial fragmentation was significantly influenced by rotenone, resulting in a lower number of solitary mitochondria per overall mitochondrial network (Fig. 7f). Rotenone also exhibited a stimulatory effect on mitochondrial network complexity (branching) (Fig. 7d). This observation may be correlated with a previous study wherein the dissipation of mitochondrial potential in non-differentiated SH-SY5Y and BJ fibroblast cells by FCCP for 3 hours resulted in a significant decrease in the average length of mitochondrial branches or rods (Valente et al. 2017; Bakare et al. 2021). In contrast to studies with FCCP on SH-SY5Y and BJ fibroblast cells, our 24-hour rotenone treatment did not lead to a significant increase in the count of rod-like mitochondria, which would indicate fragmentation.

Based on documented evidence, the most interesting finding is the stimulatory effect of diazoxide treatment on the count of sole mitochondria in differentiated cells. This phenomenon is especially pronounced after the induction of pathological conditions, where it appears to synergize with the reducing effect of rotenone on the number of solitary mitochondria rods (Fig. 7f). This finding is somewhat surprising, as the downregulation of solitary mitochondria is typically considered a positive event (Valente et al. 2017). However, based on relevant data from other authors, studies using 50 nM concentrations of rotenone on differentiated cells seem to develop pathological changes over several days, impacting cell viability and mitochondrial movement across SH-SY5Y cells (Borland et al. 2008). The concentration of rotenone used in our study can therefore be considered mild. Compensatory adaptation events occurring in living cells during exposure to rotenone could provide a hypothetical explanation for our observations, but this requires further investigation. More interestingly, the observed effect of diazoxide is the only and highly specific effect of KATP modulators-agonist on mitochondrial network remodeling observed in differentiated cells. With this in mind, we need to consider different effectors of diazoxide actions, especially since Kir 6.2 concentration is diminished in this type of SH-SY5Y. In light of recently documented diazoxide-sensitive mitochondrial KATP coded by CCDC51 gene (Paggio et al. 2019), we should take into account the possible role of this recently described potassium channel in the observed changes.

In conclusion, our data, when contextualized with existing findings, underscore the model-dependent nature of KATP channel effects. While the majority of the observed effects following treatment with KATP modulators favors the increased sensitivity of non-differentiated cells, distinctive responses are evident in differentiated SH-SY5Y cells when exposed to a KATP agonist. Despite the subdued protein expression of Kir 6.2 in differentiated cells, we noted alterations in the number of solitary mitochondria and mitochondrial potential in response to KATP modulator treatment in differentiated SH-SY5Y cells. This phenomenon warrants further investigation in future experiments and highlights the limitations of our study, particularly in the need to correlate the observed data with relevance of specific effects of intracellular KATP according to their localization. Therefore, future experiments should include a wider variety of KATP modulators to address the question of distinct effect of mitochondrial vs membrane KATP. Different differentiation protocols of SH-SY5Y as well as possible isolation of dopaminergic neurons from in vivo experiments should be performed to test potential Kir 6.2/6.1 expression under different conditions. Despite these limitations, our data provide additional support for the potential therapeutic efficacy of ATP-gated ion channels and their modulators in addressing neurodegeneration stemming from mitochondrial dysfunction. This is notably apparent in the modulation of mitochondrial morphology and concomitant alterations in energy metabolism of non-differentiated SH-SY5Y cells expressing Kir 6.2. The suitability of KATP modulators as adjunctive treatments for neurodegenerative disorders will necessitate case-specific assessments according to the unique etiopathogenesis of individual cases of neurodegeneration.