Pharmacological Rescue with SR8278, a Circadian Nuclear Receptor REV-ERBα Antagonist as a Therapy for Mood Disorders in Parkinson's Disease

Abstract

Parkinson's disease is a neurodegenerative disease characterized by progressive dopaminergic neuronal loss. Motor deficits experienced by patients with Parkinson's disease are well documented, but non-motor symptoms, including mood disorders associated with circadian disturbances, are also frequent features. One common phenomenon is "sundowning syndrome," which is characterized by the occurrence of neuropsychiatric symptoms at a specific time (dusk), causing severe quality of life challenges. This study aimed to elucidate the underlying mechanisms of sundowning syndrome in Parkinson's disease and their molecular links with the circadian clock. We demonstrated that 6-hydroxydopamine (6-OHDA)-lesioned mice, as Parkinson's disease mouse model, exhibit increased depression- and anxiety-like behaviors only at dawn (the equivalent of dusk in human). Administration of REV-ERBα antagonist, SR8278, exerted antidepressant and anxiolytic effects in a circadian time-dependent manner in 6-OHDA-lesioned mice and restored the circadian rhythm of mood-related behaviors. 6-OHDA-lesion altered DAergic-specific Rev-erbα and Nurr1 transcription, and atypical binding activities of REV-ERBα and NURR1, which are upstream nuclear receptors for the discrete tyrosine hydroxylase promoter region. SR8278 treatment restored the binding activities of REV-ERBα and NURR1 to the tyrosine hydroxylase promoter and the induction of enrichment of the R/N motif, recognized by REV-ERBα and NURR1, as revealed by ATAC-sequencing; therefore, tyrosine hydroxylase expression was elevated in the ventral tegmental area of 6-OHDA-injected mice, especially at dawn. These results indicate that REV-ERBα is a potential therapeutic target, and its antagonist, SR8278, is a potential drug for mood disorders related to circadian disturbances, namely sundowning syndrome, in Parkinson's disease.

Keywords: Circadian mood regulation; Dopaminergic neuronal loss; Nurr1; Parkinson’s disease; Rev-erbα; Sundowning syndrome.

Fig. 1

SR8278 treatment recovered mood-related behavioral deficits shown in 6-OHDA-lesioned mice. (a) Experimental scheme of behavior tests. A single injection of 6-hydroxydopamine (6-OHDA) was administrated into the left dorsal striatum. After 5 weeks post injection, mood-related behavioral tests were carried out at the indicated time of day (circadian time [CT] 22–01 vs. CT10-13). The local injection of SR8278 (20 μg/mouse) into the midbrain was performed 3 h before each behavioral test. The image was used to confirm cannula placement in the VTA by TH staining to show DA neurons in the SN and VTA (inset). Scale bar = 500 μm. (b) Changes in depression-like behaviors after SR8278 microinfusion into the midbrain of VEH- and 6-OHDA-treated mice by a forced swim test (FST) and tail suspension test (TST) (FST: two-way ANOVA, p < 0.0001 for injection group, p < 0.0001 for time, p = 0.0021 for interaction) (TST: two-way ANOVA, p < 0.0001 for injection group, p = 0.0624 for time, p = 0.0105 for interaction). (c) Alternation of anxiety-like behaviors after SR8278 administration of VEH- and 6-OHDA-treated mice by an elevated plus maze test (EPM) (duration: two-way ANOVA, p = 0.0021 for injection group, p = 0.0025 for time, p = 0.0005 for interaction) (frequency: two-way ANOVA, p = 0.0240 for injection group, p < 0.0001 for time, p = 0.0182 for interaction) (distance traveled: two-way ANOVA, p = 0.5047 for injection group, p = 0.0013 for time, p = 0.6996 for interaction) (center crossings: two-way ANOVA, p = 0.9616 for injection group; p = 0.0245 for time; p = 0.8537 for interaction). Data are presented as mean ± SEM. Sample sizes (animals) are indicated by the numbers inside bars. Newman-keuls corrected post-hoc comparisons are indicated by *p < 0.05, **p < 0.01, ***p < 0.001 and ns, non-significant

Fig. 2

SR8278 microinjection altered transcription levels of DAergic neuron-specific Rev-erbα and Nurr1 in the VTA. (a) Representative FISH images of VTA with Rev-erbα, Nurr1, and TH staining. Scale bar = 10 µm. (b) Rev-erbα and Nurr1 mRNA expression in the VTA was represented by mRNA particles in the TH + neurons divided by the counts of TH + neurons. mRNA particles in TH + neurons were analyzed by defining the region of interest (ROI). mRNA particles were assessed in the images from two sections, covering the VTA in each animal. (Rev-erbα: two-way ANOVA, p = 0.0172 for injected group, p = 0.2354 for time, p = 0.0042 for drug-by-time interaction) (Nurr1: two-way ANOVA, p = 0.4650 for injected group, p = 0.9380 for time, p = 0.0115 for drug-by-time interaction). The data were presented as mean ± SEM. Sample sizes (brain sections) are indicated by the numbers inside bars. Fisher’s LSD post-hoc comparisons are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

SR8278 restored binding activities of REV-ERBα and NURR1 and elevated TH expression at dawn. (a) REV-ERBα-bound TH promoter in midbrain lysates by ChIP-qPCR (left panel). NURR1-bound TH promoter in midbrain lysates by ChIP-qPCR (right panel). Chromatin prepared from midbrain tissues containing SN and VTA regions from two animals for each group. The binding activity was calculated as a percent of total chromatin used for immunoprecipitation (% input) and was normalized to VEH-treated control mice at CT00 (REV-ERBα: two-way ANOVA, p = 0.1166 for injected group, p = 0.0927 for time, p = 0.0023 for drug-by-time interaction) (NURR1: two-way ANOVA, p = 0.3594 for injected group, p = 0.8492 for time, p = 0.0284 for drug-by-time interaction). (b) Representative immunoblot images showing TH protein expression in the VTA derived from VEH- and 6-OHDA-lesioned mice at CT00 and CT12. Sample sizes (chromatin from two animals) are indicated by the numbers inside bars. (c) TH protein levels in VTA. After densitometric analysis, TH protein levels were normalized with actin levels (CT00: two-way ANOVA, p = 0.2508 for 6-OHDA-lesion, p = 0.0468 for SR8278 treatment, p = 0.2801 for interaction) (CT12: two-way ANOVA, p = 0.0088 for 6-OHDA-lesion, p = 0.7924 for SR8728 treatment, p = 0.3816 for interaction). Sample sizes (animals) are indicated by the numbers inside bars. The data were presented as mean ± SEM. Fisher’s LSD post-hoc comparisons are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

SR8278 microinjection to VTA altered enrichment of R/N motifs in 6-OHDA-lesioned mice only at dawn. (a) Sequence logos for REV-ERBα and NURR1 binding motifs are depicted [13]. (b) Log2 ratio (observed/expected) of ATAC-seq peaks was analyzed using HOMER. (c) Probability of R/N motifs were calculated using HOMER from called peaks and compared the motif enrichments at the indicated times for each experimental group. For each ATAC-seq data analysis, VTA regions were pooled from five individual mice (n = 5, pooled)

Fig. 5

Mood regulation and crosstalk of REV-ERBα and NURR1 in PD and SR8278-treated PD model. (a) In the normal DAergic state, the circadian nuclear receptor REV-ERBα repressed the TH gene transcription via competition with NURR1, another nuclear receptor on the same cis-element (R/N sites), inducing rhythmic TH expression [13]. Circadian oscillation of TH expression in VTA neurons resulted in the daily variation of mood-like behaviors. (b) In the PD model induced by 6-OHDA-lesion, transcription level of Rev-erbα and Nurr1 in the VTA DAergic neurons were altered at dawn, thereby inducing disappearance of rhythmic Rev-erbα transcription and disturbance of consistent Nurr1 transcription. Furthermore, 6-OHDA lesion induced atypical binding activity of REV-ERBα and NURR1 to R/N sites of TH promoter. REV-ERBα binding activity to R/N sites was increased at dawn but decreased at dusk. NURR1 binding activity was decreased at dawn without an alternation at dusk. In PD model, depression- and anxiety-like behaviors were exhibited at specific time, dawn, which time corresponds to dusk in diurnal human, characterizing the sundowning syndrome. (c) SR8278 microinjection completely restored rhythmic mood-related behaviors in 6-OHDA-lesioned mice at dawn, exhibiting the antidepressant and the anxiolytic effects in a time-dependent manner. Transcription levels of Rev-erbα and Nurr1 in the VTA DAergic neurons and binding activities of REV-ERBα and NURR1 to R/N sites were recovered by SR8278 microinjection at dawn. TH protein levels of VTA were also elevated by SR8278 microinjection at dawn. Although the binding activities of REV-ERBα and NURR1 were restored at dusk, TH expression in VTA was not recovered by SR8278 at dusk. It is noteworthy that the competitive actions of REV-ERBα and NURR1 are essential in regulating the circadian TH gene expression and mood regulation