Parkinson’s disease (PD) is the second most common degenerative disease of the central nervous system, which affects around 0.3% of the population (Raza et al., 2019). The pathological hallmarks include accumulation of filamentous, cytoplasmic inclusions consisting mainly of α-synuclein aggregations in the form of Lewy bodies (LB) in the substantia nigra of the brain, and the selective loss of dopaminergic neurons with consequently depleted dopamine levels. These deficiencies lead to motor dysfunction, with symptoms such as bradykinesia, resting tremor, muscular rigidity, and postural instability (Kalia and Lang, 2015).

PD is mainly a sporadic disorder of largely unknown etiology, which in most cases affects the elderly. The disease can result from genetic and environmental factors, although approximately 10% of all cases have a clear genetic origin, and show early manifestation, with currently at least 23 disease-segregating loci identified (Hernandez et al., 2016). Genetic and sporadic forms manifest similar clinical manifestation and indistinguishable PD hallmarks. Therefore, sporadic PD will likely benefit from studies of the molecular pathways which are impaired in familiar cases.

The identification and the study of these rare genetic PD forms identified oxidative stress, mitochondrial dysfunction, and alteration of the ubiquitin-proteasome system (UPS) and autophagy as causal factors in the pathophysiology of PD (Kalia and Lang, 2015). Most of the proteins encoded by PD genes (PINK1, Parkin and DJ1 in particular) are important for mitochondrial homeostasis and function. Accumulation of dysfunctional mitochondria can indeed contribute to cell demise in different ways. Defective mitochondria can enhance oxidative stress (Blesa et al., 2015), which exacerbates protein misfolding. They can also lead to inflammation derived from mitochondrial release of damage associated molecular patterns (mtDAMPs; Sliter et al., 2018), and impair calcium (Ca²⁺) homeostasis (Giorgi et al., 2018). PINK1, Parkin and DJ1 play a fundamental role in preventing damage originating from dysfunctional mitochondria. In particular, PINK1 and Parkin operate within the same pathway to induce the degradation of damaged mitochondria via autophagy (Narendra et al., 2010) and mitigate inflammation by repressing mitochondrial antigen presentation (Matheoud et al., 2016), while DJ1 exerts neuroprotective effects by enhancing the antioxidant defense of the cell (Kalia and Lang, 2015).

In addition to the above-mentioned primary motor function impairments, non-motor features are also frequently present in PD. Sleep disorders in particular are found in two-thirds of PD patients, and they include symptoms like insomnia, vivid dreaming, increased nocturnal activity, more fragmented sleep, REM sleep behavior disorder (RBD) and excessive daytime sleepiness (Raza et al., 2019). Because sleep behavior is controlled by circadian rhythmicity, sleep disorders can be a consequence of dysfunctional circadian clock in PD patients. Indeed, although circadian disorders are not frequently mentioned among PD symptoms, several studies in rodent PD models have shown a correlation between PD-associated neurodegenerative phenotype and circadian dysfunction (Simola et al., 2007; Hunt et al., 2022; Summa et al., 2024).

Sleep–wake cycle is controlled by the circadian clock, an internal timing system that allows adapting to and anticipating the daily changes that occur on planet Earth. It is composed by three major elements: a core oscillator, which generates the rhythmicity; inputs pathways that allow the organism to perceive the external stimuli and synchronize the core oscillator; and output pathways (represented by the circadian phenotypes), generated by the cyclic expression of clock-controlled output genes (Patke et al., 2020). At the cellular level, the circadian clock is identified by a specific subset of highly specialized neurons, called central pacemaker, which in mammals are localized in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. These neurons receive inputs from daylight, which is used to synchronize the approx. 24 h rhythm of the internal clock to the exact 24 h daily rhythm of planet Earth, to optimize the performance of the entire organism (Patke et al., 2020). At the molecular level, circadian oscillations are generated by evolutionary conserved interlocked transcriptional-translational feedback loops (TTFLs), which take approximately 24 h to complete. In TTFLs, positive elements promote the rhythmic transcription of the negative elements that inhibit in a feedback loop the activity of the positive elements. This is a self-sustained, cell autonomous mechanism generating the circadian rhythm in the SCN, which in fact persists even in the absence of light (Patke et al., 2020). In Drosophila, the circadian oscillation is based on two main interlocked TTFLs: in the primary loop, the transcription factors Clock (Clk) and cycle (cyc) control the rhythmic expression of the negative elements period (per) and timeless (tim), while the second loop results in the rhythmic expression of Clk (Hardin, 2011).

As expected, disruption of the rhythmicity can lead to the development of diseases, such as metabolic syndromes, cancer and cardiovascular diseases, and contribute to the onset of neurodegenerative diseases (Xie et al., 2019). Thus, the maintenance of proper network coordination within the clock is essential for health and well-being.

The molecular mechanism underlying the link between disturbed circadian rhythms and neurodegeneration is not known, but alteration in protein homeostasis (proteostasis) seems to be a plausible connection (Leng et al., 2019). In that respect, many studies highlighted the relevance of clock-dependent regulation of general proteostasis (Kang et al., 2009) and autophagy (Ma et al., 2011; Ryzhikov et al., 2019), and the physiological importance of sleeping as a mean to degrade proteotoxic compounds and insoluble aggregates that accumulate during the active phase (Varshavsky, 2012; Xie et al., 2013; Shokri-Kojori et al., 2018), and may induce intracellular damage.

In this study we want to exploit one well-established Drosophila model of PD that manifest abnormal circadian rhythmicity and disturbed sleep–wake cycle, to investigate the effect of enhancing proteostasis in the context of circadian and sleep disturbances. We can enhance proteostasis by down-regulating deubiquitinating enzyme (DUB) Usp14. Usp14 is a DUB, belonging to ubiquitin-specific processing (USP) family, which negatively regulate the ubiquitin-proteasome system (UPS), autophagy and mitophagy. Hence, its inhibition is predicted to both enhance the activity of the UPS, autophagy and mitophagy. Previous studies have shown that genetic down-regulation of Usp14 or its pharmacological inhibition by specific inhibitor IU1, recovered locomotor behavior of Pink1 KO flies, and it extended flies’ lifespan, presumably by enhancing protein and organelle homeostasis (Chakraborty et al., 2018). With that in mind, this work wants to test the effect of down-regulating Usp14 in Pink1 KO flies in the context of circadian rhythmicity and sleep–wake cycle.

Our results show that down-regulation of Usp14 ameliorates sleep disturbances and circadian defects that are associated to Pink1 KO flies.

Parkinson’s disease (PD) is a multifactorial devastating degenerative disease of the central nervous system, which affects around 0.3% of the population (Raza et al., 2019). PD is characterized by intracellular accumulation of α-synuclein aggregates that form the Lewy bodies (LB) in the substantia nigra of the brain, and cause the selective loss of dopaminergic neurons with consequently depleted dopamine levels (Dauer and Przedborski, 2003; Mullin and Schapira, 2015). Motor dysfunctions such as bradykinesia, resting tremor, muscular rigidity, and postural instability are the main symptoms that characterize PD (Litvan et al., 2003).

Besides motor impairments, non-motor dysfunctions are commonly present in PD patients, and they include neuropsychiatric symptoms (depression, cognitive dysfunctions and dementia; Biundo et al., 2016), autonomic symptoms (bladder disorders, orthostatic hypotension, erectile impotence; Chaudhuri and Schapira, 2009), and disturbances in sleep–wake cycle (Iranzo et al., 2006). These non-motor symptoms (NMSs) have gained increasing relevance for their impact on the quality of life of PD patients, and their contribution to institutionalization at advanced disease. They are not only common in early PD, but they can also manifest before the onset of the classical motor symptoms. Circadian and sleep disturbances in particular can precede the onset of the cognitive decline and motor symptoms by many years, suggesting that disruption of circadian rhythm might not simply be a consequence of PD but a pathogenic component of the disease that can lead to or at least contribute to neurodegeneration (Videnovic and Golombek, 2013).

The mechanistic link between altered circadian rhythms and PD is poorly understood, but proposed underlying mechanisms suggest alterations in protein and organelle homeostasis (i.e., proteostasis; Leng et al., 2019). Proteostasis is gradually lost during physiological aging. In neurodegenerative diseases, like PD, loss of proteostasis is exacerbated, and depends on the pathological decay in the proteolytic activity of the ubiquitin-proteasome and the lysosome-autophagy system, the two major degradative systems of the cell (Bennett et al., 2005; Komatsu et al., 2006). Indeed, PD is characterized by accumulation of misfolded proteins and dysfunctional mitochondria, which can be at least in part ascribed to defective mechanisms of proteostasis (Cuervo et al., 2004). Thus, promoting the activity of the UPS or autophagy is likely to be beneficial in models of PD because it enhances the degradation of misfolded proteins and dysfunctional organelles, which can be sources of oxidative stress and cellular damage (McNaught et al., 2004). In this scenario, the “proteostatic” effect of Usp14 inhibition can be a valuable therapeutic approach, due to its unique ability to both enhance the UPS (Hanna et al., 2006; Lee et al., 2010, 2016), autophagy (Xu et al., 2016; Kim et al., 2018) and mitophagy (Chakraborty et al., 2018).

We previously reported that genetic down-regulation or pharmacological inhibition of Usp14 in Pink1 and Parkin KO flies extended flies lifespan, and rescued the locomotor phenotype associated with these flies (Chakraborty et al., 2018), presumably by promoting proteostasis. In the current work we wanted to extend these findings, and address the potential beneficial effect of Usp14 inhibition in the context of circadian dysfunction in Pink1 KO flies modeling PD.

Pink1 mutants develop very obvious circadian defects. In particular, Julienne H. and co-authors showed a decrease in total activity in Pink1 KO flies, and a significant reduction in rhythmicity. Among those flies that remained rhythmic, authors did not observe a significant difference in the circadian period (Julienne et al., 2017). A parallel study reported a trend toward a decrease in total activity of Pink1 KO flies, while a significant decrease in total activity was shown when Pink1 was specifically down-regulated in elav expressing cells. In this study, Pink1 KD flies displayed increased daytime sleep, whereas night-time sleep was not affected (Doktor et al., 2019). In support of these studies, we also observed defective circadian behavior in Pink1^B9 mutant (Pink KO) flies. In LD conditions, Pink1 KO flies are less active during the day, with reduced ability to anticipate morning and evening clues. The percentage of rhythmic flies was significantly reduced in Pink1^B9 mutant flies. Among those flies that remained rhythmic, the circadian period was significantly lengthened. We also found that Pink1^B9 flies sleep significantly more during the night and during the day compared to wild type. The quality of their sleep appeared to be different compared to wild type flies. In particular, Pink1 KO displayed fewer but longer episodes of sleep during the night and during the day (i.e., longer “siesta”).

Our results showed that down-regulation of Usp14 completely rescued the circadian phenotype of Pink1 KO mutants, and the sleep defects of these flies, while Usp14 down-regulation alone in wild type background did not seem to affect the circadian clock, with some mild effect that was readable only in clock-relevant neurons.

The molecular mechanism by which Usp14 ameliorates PD circadian symptoms in flies is unknown. It may be direct, by altering protein stability of core components of the circadian clock, or indirect. It is known that in mammals Usp14 affects the circadian period by slowing down the degradation of clock protein Period (PER; D'Alessandro et al., 2017). However, the mild effect of Usp14 down-regulation on the circadian phenotype of wild type flies suggests that protein level of core components of the clock is not significantly affected by Usp14 deubiquitinating activity, although this circumstance cannot be excluded. Another possibility is that Usp14 rescues circadian defects of Pink1 KO flies by an indirect mechanism, for example by promoting mechanisms of mitochondrial quality control that relies on mitochondrial dynamics. It is known that promotion of mitochondrial fission rescues the aberrant phenotype and circadian defects associated with mice lacking a functional clock (Jacobi et al., 2015), and recovers disrupted circadian oscillation of oxidative phosphorylation and mitochondrial dysfunction of cells with specific ablation of clock gene BMAL1 (Jacobi et al., 2015; Li et al., 2020). Intriguingly, cells lacking mitochondrial fission do not display circadian oscillation of core component of the circadian clock (Schmitt et al., 2018), suggesting that dysregulation in mitochondrial dynamics likely feedbacks to the circadian clockwork. Because Usp14 down-regulation promotes mitochondrial fission and mitophagy (Chakraborty et al., 2018), it is tempting to speculate that the normalization of the circadian defects may arise from the pro-fission effect of Usp14 down-regulation, which is key to mitochondrial quality control.

Clinical diagnosis of PD is mainly based on the presence of motor symptoms, which usually appear when more than 50% of dopaminergic neurons have already been lost. Since the diagnosis occurs too late for neuroprotective treatments to work, it is important to find a way to diagnose the disease before the appearance of motor symptoms, and implement a therapy at the premotor phase to prevent or delay the development and progression of disease (Kalia and Lang, 2015). In this respect, sleep disturbances, as well as non-motor symptoms in general, may offer a pre-symptomatic window for the treatment of this disease, allowing earlier intervention (Julienne et al., 2017). In this premotor phase, the pathogenic process of PD is presumed to be underway, involving regions of the peripheral and central nervous system that are distinct from the dopaminergic neurons of the substantia nigra pars compacta (SNpc). At present we do not know whether improving sleep and circadian behavior early on in PD patients may delay or even prevent the progression of the diseases. Circadian and locomotor dysfunction appears early in Pink1 KO flies, and they are tested when flies are relatively young (3 to 6 days). In this respect, the fly model does not fully recapitulate the progression of the disease in human patients, where, particularly in the sporadic forms, there is a time gap between the appearance of the locomotor and the circadian-related symptoms. Thus, we cannot address this relevant hypothesis in flies, but it could be in principle addressed in mice. There are at least two murine models, the 6-hydroxydopamine (6-OHDA) model (Simola et al., 2007) and the MitoPark mouse (Galter et al., 2010), where circadian rhythm and sleep behavior have been fully characterized. Both models show a reduction of locomotor activity, accompanied by a severe disruption of circadian rhythm in constant darkness, which are indicative of parallel sleep dysfunctions (Fifel and Cooper, 2014; Masini et al., 2017).

Importantly, highly specific and potent small-molecule inhibitors of Usp14 are available, which are orally bioavailable, can cross the blood–brain barrier (BBB), and are well-tolerated in murine models. Usp14 inhibitors (IU1 and IU1-47) were originally developed some years ago (Lee et al., 2010; Boselli et al., 2017), and continue to be optimized from the original backbone. Given their high specificity (Wang et al., 2018), the development of therapeutic approaches targeting Usp14 seems to be a feasible therapeutic avenue.

In summary, in this work we strengthened Drosophila as animal model for investigating sleep and circadian symptoms of PD, and extended the evidences on the protective effect of Usp14 down-regulation in the context of circadian and sleep disturbances.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The manuscript presents research on animals that do not require ethical approval for their study.

MF: Data curation, Writing – review & editing, Investigation, Methodology, Visualization. SM: Investigation, Methodology, Writing – review & editing, Formal analysis, Supervision. GB: Formal analysis, Investigation, Writing – review & editing. MZ: Formal analysis, Writing – review & editing. GM: Formal analysis, Writing – review & editing, Conceptualization, Data curation, Methodology, Supervision. EZ: Conceptualization, Data curation, Formal analysis, Supervision, Writing – review & editing, Funding acquisition, Resources, Writing – original draft.