The effect of formoterol on mitochondrial function has been validated in various diseases, including traumatic brain injury[25], spinal cord injury[26], diabetic kidney disease[27], and acute kidney injury, with those studies providing compelling evidence in support of the use of β2AR ligands for therapeutic mitochondrial biogenesis; however, the machinery underlying the mitochondrial dynamics of fusion/fission and movement remains unclear in PD, and their therapeutic efficacy is controversial. In particular, abnormalities in mitochondrial dynamic processes, specifically in fusion and fission, play a crucial role in the pathogenesis of PD[13]. This study initially revealed that formoterol treatment improved cell proliferation and neural cell activity and had a neuroprotective effect against ROS-induced cell death. Mitochondrial dysfunction was also comprehensively restored, including the dynamic imbalance of fusion/fission, mitochondrial movement, and mobility. The related machinery may involve the activation of the ERK and the inhibition of the Akt signaling cascades downstream of B2AR, thus restoring mitochondrial fission and decreasing persistent hyperfusion. This contributes to the segregation of healthy mitochondria for proper distribution during cell division and mitochondrial transport, as our findings showed that the facilitation of mitochondrial anterograde movement by formoterol normalized mitochondrial distribution in UQCRC-mutant neural cells.
Although mitochondrial function and dynamics are generally interrelated, in certain cases the interconnection between mitochondrial dynamics and vital function can be dissociated[28]. The inhibition of mitochondrial respiration and mitochondrial ATP production using sublethal concentrations of mitochondrial complex I (CI) and CIII inhibitors did not trigger mitochondrial fragmentation, in contrast to CV inhibitors[29]. This revealed that CIII may play an energy-independent role in the regulation of the mitochondrial dynamic balance. A recent study showed that, in UQCRC2-mutant (c.665G > C; p.Gly222Ala) fibroblasts derived from patients with severe encephalomyopathy, a significant mitochondrial fragment was accompanying by a high degree of mitochondrial branching[30]. It is well known that UQCRC1 and UQCRC2 are both subunits of CIII that play essential roles in the structure and function of CIII. Mutations or disruptions in these subunits can lead to mitochondrial dysfunction and various related disorders[16, 30]. We also found a notably increased reticular network of mitochondria in UQCRC1-mutant neural cells; however, unlike that observed in UQCRC2-mutant cells[30], the proportion of fragmented mitochondria was relatively decreased. Moreover, a notable increase in Mfn2, but not OPA1, and a decrease in Drp1 were observed in UQCRC1-mutant cells, whereas Mfn2 and OPA1 remained unaffected and Drp1 was significantly upregulated[30]. This suggests that UQCRC1 mutation has a more significant impact on disrupting the mitochondrial dynamics balance toward fusion, to adapt to stressful conditions. This regulation could be reversed by reducing mitochondrial stress through formoterol treatment, thereby restoring a balance between mitochondrial fusion and fission and preventing excessive mitochondrial fusion and aggregation. In fact, excessive mitochondrial hyperfusion impairs mitochondrial distribution and turnover, thus inducing locomotor defects in Drosophila models of Charcot–Marie–Tooth disease type 2A neuropathy[31]. Prolonged elongation of mitochondria has been demonstrated to impair mitophagy, which consequently affects the clearance of dysfunctional mitochondria and diminishes mitochondrial function[32, 33]. Moreover, the maintenance of elongated mitochondria leads to the disruption of its distribution and transport in cells[28, 34], as evidenced in the present study. In addition, abnormally elongated mitochondria also interfere with the interactions with other cellular structures, thus potentially affecting cellular signaling and responses[28].
Formoterol treatment led to the comprehensive restoration of mitochondrial function, including mitochondrial respiration and CIII activity and biogenesis, with the exception of ATP production. Based on our findings, we suggest that this could be related to the increase in mitochondrial biogenesis, for the generation of new mitochondria, which can increase the overall respiratory capacity of cells. Furthermore, this study further found that increasing the availability of certain metabolic substrates via cellular signaling and regulatory mechanisms, such as the ERK pathway, may lead to an increase in BR without necessarily a direct impact on ATP synthesis[35, 36]. This supports our finding that the increase in the availability of mitochondrial substrates, i.e., glutamate and malate, can be considered as an influencing factor. Conversely, formoterol activates the activated cyclic AMP-PKA pathway, which is a β2AR downstream effector pathway that has been shown to modulate the regulation of mitochondrial metabolism and function[37]. Interestingly, we found that formoterol treatment did not further enhance the already high performance of PKA activity in mutant cells compared with WT cells. This suggests that the effects of formoterol on mitochondrial function may vary according to various factors, such as cell type, dosage, treatment duration, and specific experimental conditions[27].
Drp1 is a key regulator of mitochondrial fission that can be phosphorylated at different sites by distinct signaling transduction guided in context, to adjust the process of mitochondrial fission. For instance, phosphorylation of Drp1 S616 enhances its GTPase activity, leading to increased mitochondrial fission. Conversely, phosphorylation of Drp1 Ser637 inhibits this process[38, 39]. We found that formoterol treatment increased Drp-1 activity via phosphorylated Drp1 S616 and dephosphorylated Drp-1 S637, to provide a fine-tuned control mechanism for mitochondrial fission. Consequently, extensive elongation of the mitochondrial network was decreased, restoring a balanced pattern between mitochondrial fission and fusion. Recently, a study reported the contribution of the Akt-1 pathway to the inhibition of mitochondrial fission through the parallel regulation of dephosphorylated Drp1 S616 and phosphorylated Drp1 S637. Conversely, the MEK1–ERK pathway was involved in promoting mitochondrial fission through the phosphorylation of Drp1 S616 and dephosphorylation of Drp1S637[38]. The two axes of the Akt1–Drp1 and MEK1–ERK-Drp1 pathways can be switched to remodel the mitochondrial dynamics in somatic cell reprogramming[38]. This reflects our finding that UQCRC1-mutant cells, which have a deficiency in mitochondrial fission, displayed a lower ERK signaling and a higher Akt activity compared with the WT cells. Formoterol certainly adjusted the dynamic toward fission through the parallel regulation of activating ERK signaling for the phosphorylation of Drp-1 S616 and inhibiting Akt signaling for the phosphorylation of Drp-1 S637. Furthermore, mutant cells exhibited increased levels of Mfn2 proteins, in contrast to Drp-1. The physiological levels of Mfn2 expression are strongly correlated with the Akt signaling pathway, to promote mitochondrial fusion[40]. Therefore, the literature mentioned above consistently emphasizes that the overactivation of the Akt pathway, as opposed to ERK signals, causes an imbalance in mitochondrial dynamics, thus favoring fusion over fission. This disruption in the dynamic balance of mitochondria can have significant implications for cellular functions and may contribute to various cellular processes and diseases.
Several of the known genes associated with the familial forms of PD are involved in the regulation of mitochondrial function, including parkin and Pink-1[41]. The downregulation of Pink-1 affects the mitochondrial fusion–fission machinery and sensitizes mitochondria to neurotoxins in dopaminergic cells[42]. Pink-1 not only phosphorylates Drp-1 S616 to activate mitochondrial fission[43], but also phosphorylates Mfn2 to promote parkin recruitment for mitophagy, which is a selective process that removes damaged mitochondria[44]. Loss of Pink-1 impairs mitochondrial function, leading to mitochondrial dysfunction, increases in oxidative stress, and a compromised cellular energy production[45]. This is in line with our observation of reduced Pink-1 expression in mutant cells. Interestingly, in cells treated with formoterol, both full-length Pink-1 and its 52-kDa cleaved form[46] were upregulated, whereas the levels of parkin and Mfn2 were significantly decreased. Thus, we suggest that the formoterol-induced upregulation of Pink-1, with consequent mitochondrial fission, does not active mitophagy. In fact, Pink-1 regulates mitophagy-independent mitochondrial fission by phosphorylating Drp-1 S616[43]. Moreover, increasing the levels of cytosolic cleaved Pink-1 and ablating Mfn2 also repressed parkin translocation to mitochondria for mitophagy[44, 47]. Conversely, although the role of cleaved Pink-1 in neuronal functions remains poorly understood, in healthy mitochondria, Pink-1 is cleaved at the inner mitochondrial membrane and is retro-translocated to the cytosol, where it exerts extra-mitochondrial functions that are important for neuronal development, neuronal survival, synaptogenesis, and plasticity; these are crucial implications for PD[48, 49]. Thus, we suggest that restoration of Pink-1 could play a multifaceted role in supporting neuronal function in the context of the benefits of formoterol.
Formoterol has been shown to decrease the glucose-induced imbalance in mitochondrial dynamics and restore mitochondrial homeostasis in the context of renal proximal tubule cells under diabetic conditions[50]. A similar regulatory role of formoterol was found by us in the treatment of UQCRC1-mutant cells resulting in PD, although the specific mechanisms underlying the regulation of mitochondrial homeostasis appear to be dependent on various cellular contexts. Moreover, our findings provide further evidence of the beneficial effects of formoterol, as it significantly promoted anterograde mitochondrial transport and increased mitochondrial mobility. Several studies have explored strategies aimed at enhancing anterograde mitochondrial transport as a means of neuroprotection, to manage mitochondrial-dysfunction-related neurodegenerative disorders[51–53]. Because the promotion of mitochondrial anterograde movement facilitates the transport of mitochondria toward axon terminals, to support the energy demands required for synaptic transmission and neuronal signaling[52, 53], maintain mitochondrial health via the overall quality control of mitochondria within neurons[53], and ensure proper neuronal functions, such as calcium regulation, excitability, and neurotransmission[51, 52]. However, the causes of the reduction in anterograde mitochondrial axonal transport and mitochondrial mobility caused by UQCRC1 mutations, whereas retrograde movement is unaffected, remain unclear. Based on limited findings, we suggest that sustained mitochondrial hyperfusion could hinder mitochondrial transportation by modifying the size, shape, and distribution of mitochondria. Mitochondrial hyperfusion has been indicated to disrupt the interactions between mitochondria and motor proteins, affecting their proper engagement and transport along the cytoskeleton during stress promotion[54]. The entanglement and close proximity of fused mitochondria could restrict their movement along microtubules, thereby reducing their mobility to distribute and reach specific cellular regions [54]. In contrast, studies have shown that Pink1 forms a multiprotein complex with Miro and Milton, thus linking Pink1 function to regulate mitochondrial movement in axons [55, 56], and its effect on mitochondrial motility is direct, rather than a secondary effect of changes in mitochondrial length[56]. Moreover, the localization of cleaved Pink-1 to the cytosol has been shown to enhance the anterograde-mediated trafficking of mitochondria in neurites (both in dendrites and axons) via the phosphorylation of Miro2[48, 57]. Therefore, the regulatory role of Pink-1 in mitochondrial transportation is worthy of further investigation.