Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world, with approximately 6.1 million people affected worldwide in 2016. PD occurs mainly in older age groups, with a prevalence of about 0.5–1% in people aged 65–69 years, rising to 1–3% in people aged 80 years and older, and with a higher prevalence in men than in women (Tanner and Goldman, 1996; GBD 2016 Parkinson’s Disease Collaborators, 2018). With the aging of the population, the prevalence and incidence of PD are projected to increase by 30% by 2030, posing a heavy burden on social development (Chen et al., 2001).

Patients with PD often exhibit motor and non-motor symptoms. Motor symptoms such as bradykinesia, rigidity, resting tremor, postural instability; and non-motor symptoms include loss of smell, sleep disturbance, autonomic dysfunction, psychological disorders, cognitive impairment, etc. (Kouli et al., 2018; Armstrong and Okun, 2020). The exact cause of PD is still unknown, and studies have shown that its occurrence may be related to various factors such as genetics, environment, and lifestyle. The main pathological features of PD are abnormal aggregate of alpha-synuclein (α-syn) to form Lewy bodies and progressive loss of dopaminergic neurons in the substantia nigra compacta. Mitochondrial dysfunction is also thought to play a vital role in the development of PD pathology. Mitochondria produce ATP through the process of oxidative phosphorylation, which is the primary source of intracellular energy production, and impaired mitochondrial function in PD patients leads to a reduction in energy production, which negatively affects neuronal function and survival. In addition, mitochondria are the main source of oxidative stress, and the generation of reactive oxygen radicals can cause damage to cellular components (such as proteins, lipids, and DNA), and mitochondrial dysfunction in PD exacerbates oxidative stress, further triggering cytotoxic and inflammatory responses (Grunewald et al., 2019; Monzio Compagnoni et al., 2020; Malpartida et al., 2021). When mitochondria are damaged, cells degrade the damaged mitochondria through selective autophagy (i.e., mitophagy) to regulate cellular homeostasis.

There is a close relationship between α-syn and mitophagy, which plays an essential role in the pathogenesis of PD. Mitophagy can remove abnormally aggregated α-syn, and enhanced mitophagy reduces α-syn aggregate, thereby attenuating the pathological progression of PD (Picca et al., 2021). Moreover, the abnormal aggregate of α-syn can interfere with the normal function of mitophagy-associated proteins (e.g., PINK1, Parkin), thus affecting the mitophagy process (Minami et al., 2015).

Currently, PD treatment can only relieve patients’ symptoms rather than cure them. Therefore, exploring the cause of the disease and clarifying the pathological process are crucial for furthering understand PD and finding appropriate treatments. Further exploration of the roles played by α-syn and mitophagy in the pathology of PD, and their interactions, has become an important starting point to clarify the pathogenesis of PD.

Recent studies have revealed a close relationship between phase separation and PD. Such as oil drops in water, the process by which different components of a liquid environment are separated to form two or more distinct phases under certain conditions due to differences in their biophysical properties is known as liquid–liquid phase separation (LLPS). In living cells, biomolecules (proteins or nucleic acids) are separated by LLPS into liquid-like, non-membranous bodies (called phases, also known as biomolecular condensate) with specific functions, and there are distinct interfaces between the different phases to form separate compartments isolated from the external environment, ensuring that different biochemical reactions take place in time- and space-constrained compartments (Wang and Zhang, 2019; Gouveia et al., 2022). An increasing number of studies have shown that LLPS is involved in the formation of intracellular membraneless organelles, such as P granules (Brangwynne et al., 2009), nucleolus (Brangwynne et al., 2011), stress granule proteins (SG) (Molliex et al., 2015) etc. LLPS of biomacromolecules (proteins or nucleic acids) has become an important mechanism to assist cellular functions. In a solution containing a biomolecule, biomolecules and solvent molecules tend to be evenly distributed to maintain the maximum entropy value and the lowest free energy (Alberti et al., 2019). When the concentration of biomacromolecules gradually increases, the biomacromolecules gradually aggregate and self-assemble to form a concentrated phase due to multivalent interactions between molecules, especially weak interactions. In this case, their reduced entropy value is compensated by the additional molecular interactions in the concentrated and diluted phases (Zhang et al., 2020). The possession of intrinsically disordered regions (IDR) and low-complexity domains (LCD) is one of the characteristics of proteins capable of LLPS, with IDR lacking a stable conformation that contributes to the involvement of intermolecular interactions. LCD is characterized by amino acid bias and/or repetitive linear motifs, a feature often associated with structural disorders of proteins (Molliex et al., 2015).

Biomolecules form condensate through LLPS, the condensate is not static after its formation and will change as the external environment changes and the internal structure is adjusted (Wang and Zhang, 2019). In the initial stage of LLPS to form a condensate, the mutual gravitational force between the liquid molecules is not sufficient to hold the entire liquid phase clumps together, and these condensates float in solution in the form of a liquid with a high degree of mobility. With the passage of time or under the action of various influencing factors, the intermolecular gravitational force inside the condensate gradually increases, and the structure of the droplet gradually becomes more organized. This ordered structural state is called gel-like, gel-like has a certain degree of elasticity and solid nature, but still has a certain degree of mobility. Based on gel-like formation, further interactions can lead to re-aggregate of the gel to form fibrous aggregate (Alberti et al., 2019). The size, formation rate, and biophysical properties of phase-separated protein condensate are important for their realization of different cellular functions, and such properties change when influenced by different factors, such as the transition from a highly fluid liquid state to a hydrogel state and eventually to a solid-like condensate (Banani et al., 2017; Alberti et al., 2019). In addition, multiple factors (e.g., environmental changes, changes in the protein itself, protein–protein interactions, etc.) have been found to affect the physiological functions of proteins/RNA by influencing the LLPS process of biomolecules (Ray et al., 2020; Poudyal et al., 2022; Huang et al., 2022a).

In addition, LLPS also plays a vital role in the study of human pluripotent stem cells (hPSCs), where many key transcription factors and RNA-binding proteins can undergo phase separation phenomena to form condensate that influences the expression of specific genes and cell fate decisions and is involved in the regulation of the process that maintains the stemness and self-renewal capacity of hPSCs (Kim, 2021; Lim and Meshorer, 2021).

Recently, we found that multiple neurodegenerative disease-associated proteins undergo the biophysical process of LLPS, such as Tau protein in Alzheimer’s disease (AD) (Ambadipudi et al., 2017; Wegmann et al., 2018; Kanaan et al., 2020; Wen et al., 2021), α-syn in PD (Hardenberg et al., 2020; Ray et al., 2020), FUS (Patel et al., 2015; Zbinden et al., 2020) and TDP-43 (Conicella et al., 2016; Liu and Fang, 2019) associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), etc. The current focus is mainly on α-syn and phase separation processes in mitophagy. LLPS is observed to occur in the early stages of α-syn aberrant aggregate (Elbaum-Garfinkle, 2019; Ray et al., 2020), the formation of biomolecular condensates by α-syn phase transition is closely related to the pathogenesis of PD. In addition, LLPS has been found to be involved in the regulation of mitophagy processes (Noda et al., 2020; Yamasaki et al., 2020; Peng P. H. et al., 2021; Xing et al., 2021; Brodin et al., 2022). LLPS in α-syn and mitophagy are discussed further below. Therefore, it is important to observe and study the role of LLPS in α-syn abnormal aggregate in PD and its role in regulating mitophagy for our further understanding of the pathogenesis of PD.

This review generalizes the occurrence, development, and related influencing factors of α-syn LLPS, a key pathological protein in PD, and the research progress of LLPS in PINK1-Parkin-mediated mitophagy, starting from the mechanism of the role of biomolecular condensates in the process of α-syn aggregate and mitophagy in PD, and discusses its importance in the development of PD pathology, hoping to provide new ideas and methods for the pathological research of PD.

α-syn misfolding and aggregate is a key target for PD treatment. Therefore, observing the role of α-syn LLPS in aggregate is crucial for us to clarify the pathogenesis of PD further (Giampa et al., 2021; Gadhe et al., 2022; Li et al., 2022). α-syn is a naturally unfolded protein consisting of 140 amino acids, which is abundantly present in presynaptic nerve endings, and consists of three structural domains: a positively charged amphiphilic N-terminal domain (residues 1–60), which interacts with the membrane; a hydrophobic non-amyloid beta component (NAC) domain (residues 61–95), which is involved in fiber formation and aggregate; and a negatively charged acidic C-terminal domain (residues 96–140), associated with α-syn nuclear localization and involved in the interaction of α-syn with metal ions, ligands and other proteins (Uversky and Eliezer, 2009; Poudyal et al., 2022). It was found that the LCD in the N-terminal and NAC domains of α-syn are the key factors driving the phase separation of α-syn, but exactly which residues are involved is still unknown.

In vitro, expression of purified α-syn undergoes LLPS and occurs before α-syn aggregate. The formation of liquid-like condensates of α-syn in the presence of 10% polyethylene glycol (PEG)-8,000 at a concentration of ≥200 μm was observed by differential interference contrast microscope (DIC), which was further confirmed by light scattering and fluorescence imaging using fluorescein isothiocyanate (FITC)-labeled α-Syn (10% labeled). Over time, α-syn condensates undergo an abnormal phase transition from liquid (day 2) to gel (day 5) to solid-state (day 30), with reduced mobility and migration capacity, eventually giving rise to amyloid fibril aggregate. Also, α-syn condensates were observed to appear in the cellular model and transform into perinuclear aggregates (Ray et al., 2020). This phenomenon was also observed in nematodes, when yellow fluorescent protein (YFP)-tagged human α-syn protein was stably expressed in nematode body wall muscle cells, the emergence of condensates in nematode adults was observed by high-resolution fluorescence lifetime imaging microscopy (FLIM), and the liquid state (days 1–11) was gradually transformed into a starch-rich hydrogel with the nature of Louisianian vesicles as nematodes grew (days 13–15) (Hardenberg et al., 2021).

β-syn, γ -syn is a member of the same synaptic nuclear protein family as α-syn and is also closely associated with the pathogenesis of PD. β-syn is a 134 amino acid protein with extensive synaptic co-localization with α-syn. It cannot aggregate alone due to the lack of hydrophobic residues 73–83 in its NAC region. β-syn can also play a neuroprotective role by inhibiting α-syn aggregate and fiber formation (Kahle et al., 2000; Uversky et al., 2002; Sharma et al., 2020), V70M and P123H alter the structure and amyloidosis of β-syn, which accelerates the aggregate of β-syn (Ohtake et al., 2004). γ-syn is structurally similar to α-syn and can form fibers alone, but its formation rate is much slower than that of α-syn.

Interactions between members of the synaptic nucleoprotein family have been widely discovered, but whether β-syn, γ -syn is involved in the regulation of α-syn LLPS is currently unknown. Observing the effect of β-syn, γ-syn on α-syn condensate formation by LLPS may provide us with new insights to study the interactions between synaptic nucleoproteins and prevent abnormal aggregate of α-syn.

Cellular homeostasis is an important basis for the organism to maintain normal physiological activities. Therefore, maintaining cellular homeostasis is of great significance for disease prevention and treatment. Currently, cells maintain homeostasis by removing damaged components through two main pathways: (1) Ubiquitin-proteasome system (UPS): degrades short-lived proteins in cells. (2) Autophagy-lysosome pathway (ALP): digests long-lived proteins in cells and participates in the autophagy of abnormal organelles (Nijholt et al., 2011). These two systems are synergistically involved in maintaining the normal function of neurons, and when they become dysfunctional, they trigger the development of neurodegenerative diseases, such as PD.

Cellular autophagy plays an essential role in maintaining cellular metabolic homeostasis and involves a series of processes such as double membrane formation, extension, vesicle maturation (called autophagosomes), and translocation of target cargo to lysosomes (Glick et al., 2010; Ghavami et al., 2014). Mitochondria are important double-membrane organelles in the cell and play a role in fundamental processes of cellular activity such as ATP production, calcium signaling, and iron homeostasis (Raffaello et al., 2016; Spinelli and Haigis, 2018). Mitophagy is the process by which damaged and aged mitochondria are delivered to lysosomes for degradation via the autophagic pathway for mitochondrial quality and quantity regulation (Pickles et al., 2018; Onishi et al., 2021). Mitophagy is a crucial way for cells to prevent damaged mitochondria accumulation and perform mitochondrial quality control (Liu et al., 2019).

Narendra et al. (2008) first revealed PINK1-Parkin-mediated mitophagy and demonstrated that it is one of the key pathways of mitophagy (Nardin et al., 2016; Kumar et al., 2017). PINK1 is a mitochondrial targeting protein that enters mitochondria under physiological conditions via the translocase of the outer membrane (TOM) complex on the outer mitochondrial membrane (OMM) and the inner membrane translocase (TIM) 23 complex on the translocase of the inner membrane (IMM) (Pickrell and Youle, 2015; Sekine and Youle, 2018). When PD induced mitophagy occurs, mitochondria undergo depolarization, PINK1 accumulates on the OMM, and the S228 and S402 sites undergo autophosphorylation while phosphorylating the S62 site of the Parkin Ubl structural domain, recruiting and activating the E3 ubiquitin ligase Parkin, which in turn ubiquitinates mitochondrial outer membrane-associated proteins and recruits autophagy receptors (e.g., p62/SQSTM1, etc.), isolate and translocate damaged mitochondria for degradation via the autophagy-lysosome pathway, and mediate mitophagy (Narendra et al., 2008; Matsuda et al., 2010; Ziviani et al., 2010; Heo et al., 2015; Lazarou et al., 2015; Wauer et al., 2015; Han et al., 2020). Dysregulation of mitophagy may contribute to the neurodegenerative pathogenesis of PD.

Mitophagy plays an important role in PD. Abnormal mitochondrial function and damage are present in patients with PD, mitophagy can remove the damaged mitochondria and reduces mitochondrial dysfunction and mitochondria-associated cytotoxicity (Malpartida et al., 2021). This helps maintain the balance of energy metabolism within the cell and the healthy state of the mitochondria. Furthermore, mitochondria are one of the major intracellular sources of oxidative stress, and when mitochondrial function is impaired, it leads to excessive oxidative stress and mitochondrial DNA damage (Lizama and Chu, 2021). Mitophagy reduces oxidative stress and mitochondrial DNA damage, thereby protecting neurons from damage. Therefore, mitophagy is considered a potential therapeutic target for PD.

Recently, LLPS has also been observed in mitophagy (Hollenstein and Kraft, 2020; Noda et al., 2020; Darling and Shorter, 2021; Hayashi et al., 2021; Zhang, 2022), Parkin, autophagy receptor p62/SQSTM1, TFEB, and several other biomacromolecules play important roles in mitophagy (Figure 2). However, the mechanisms by which these biomolecules undergo selective autophagy, how their biophysical properties affect their physiological functions, and whether they are associated with susceptibility to autophagic degradation remain unclear. Here we highlight recent studies on phase separation in PINK-Parkin-mediated mitophagy and explore the role and mechanisms of biomolecular condensates in regulating PINK-Parkin-mediated selective autophagy (Table 1).