Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neurodegeneration. In patients with PD, neurodegeneration is correlated with the pathological accumulation of α-synuclein (α-syn) aggregates (Goedert et al., 2013). The aggregation of α-syn is regulated by a series of specific factors, including posttranslational modifications, such as kinase-mediated phosphorylation, and interactions with the intracellular machinery, such as organelle components or molecular chaperones (Barnham et al., 2004, Lashuel et al., 2013, Mahul-Mellier et al., 2014). Studies have shown that chemical modification of α-syn affects its interactions with chaperones and other target proteins. Inhibition of the interaction of α-syn with the chaperones HSC70 and HSP90 by regulating α-syn phosphorylation triggers marked relocalization of α-syn to the mitochondria (Burmann et al., 2020). This localization of α-syn to the mitochondria disrupts the mitochondrial membrane, and the impairment of mitochondrial function may exacerbate the progression of PD (Park et al., 2018, Reeve et al., 2018). However, the mechanisms underlying α-syn phosphorylation and α-syn-mediated neuronal cell injury remain elusive.

In the healthy brain, α-syn is mainly present in its unphosphorylated state. However, in patients with PD, more than 90% of abnormally aggregated α-syn proteins are phosphorylated at Ser129 (Kawahata et al., 2022). The phosphorylation of α-syn, particularly at Ser129, is considered to be pathologically significant. The phosphorylation of α-syn at Ser129 inhibits its binding to the cellular membrane. As its binding to the plasma membrane decreases, phosphorylated α-syn preferentially localizes to mitochondria and causes mitochondrial dysfunction (Di Maio et al., 2016, Wang et al., 2019). The mechanisms underlying the mitochondrial damage induced by phosphorylated α-syn warrant further study.

One of the key pathological features of neurons in PD is the global dysregulation of Ca2+ homeostasis (Zaichick et al., 2017). The mitochondrial damage that occurs in PD is reportedly associated with mitochondrial calcium overload (Amorim Neto et al., 2022). Excessive mitochondrial uptake of Ca2+ or impaired efflux of Ca2+ results in the production of reactive oxygen species (ROS) and disruption of the membrane potential; these phenomena cause neuronal cell death, which is an important indicator of several different neurological disorders, including PD (Luongo et al., 2017, Reynolds and Hastings, 1995). Studies have revealed that impairments in mitochondrial biogenesis and Ca2+ buffering may precede the development of PD pathology (Reynolds and Hastings, 1995). However, several studies have shown that α-syn aggregates can trigger a significant increase in the mitochondrial Ca2+ levels (Ganjam et al., 2019). The network regulating α-syn phosphorylation and mitochondrial calcium overload remains unclear.

Mitochondrial calcium ([Ca2+]m) influx is mainly mediated by the mitochondrial calcium uniporter (MCU) complex in the mitochondrial membrane (Alevriadou et al., 2021, Tanwar et al., 2021). The MCU complex consists of four MCU subunits and essential MCU regulators. The MCU regulators, including mitochondrial calcium uptake 1/2 (MICU1/2) and MCU dominant-negative β-subunit (MCUb), interact with the MCU subunits. Excessive activation of MCU during the process of mitochondrial calcium uptake induces [Ca2+]m overload and mitochondrial dysfunction (Wu et al., 2019). Previous studies have revealed that targeting MCU inhibits [Ca2+]m uptake, ameliorates calcium overload and improves mitochondrial function in the endothelium (Li et al., 2020, Li et al., 2021). However, whether α-syn regulates mitochondrial calcium overload and mitochondrial function via the MCU complex in PD has not been fully explored. Although multiple proteins or processes can regulate the mitochondrial calcium levels, MCU is a major pathway of mitochondrial calcium influx depending on the concentration of extramitochondrial Ca2+ (Garg et al., 2021). Mitochondrial Ca2+ uptake via MCU is an essential process regulating mitochondrial metabolism (Dong et al., 2022). Notably, studies have confirmed that pretreatment with an MCU inhibitor can reduce the α-synuclein-related pathology in α-syn-preformed fibril-treated neurons (Apicco et al., 2021), hence the interaction between alpha-syn and MCU is noteworthy.

In this study, we aimed to identify the role of phosphokinase and the pathological effects of α-syn phosphorylation on mitochondrial calcium influx and mitochondrial function in PD. We found that calcium/calmodulin-dependent serine protein kinase (CASK) is a phosphokinase that phosphorylates α-syn at Ser129. The pathological effect of CASK on mitochondrial dynamics and MCU complex-related [Ca2+]m homeostasis was explored using PD models. Activation of the MCU complex via α-syn phosphorylation is the core mechanism underlying CASK-related mitochondrial calcium overload and mitochondrial damage in PD. In summary, this study investigated the pathological effect of CASK and explored the possible involvement of MCU and mitochondrial calcium overload to determine the potential of CASK as a target in PD treatment.