Circadian rhythm in neurodegenerative disease: the role of RNA modifications and potential application of RNA-based therapeutics

Circadian rhythms

The circadian rhythms function as an intrinsic biological clock that orchestrates various aspects of life processes, from genetic activity and cellular metabolism to overall behavioral patterns. These rhythms, constituting roughly 24-h cycles in the physiological functions of most organisms, are internally generated and prone to external influences. A typical circadian cycle shows characteristics such as being self-sustained, rhythmic, synchronized, and measurable^[18]. These rhythms are self-sustaining, maintaining a consistent oscillation independent of external cues, such as light-dark cycles, indicating the presence of an inherent timekeeping mechanism. The rhythmic quality stems from the consistent 24-h cycle of circadian processes. Additionally, circadian cycles can synchronize with external factors; hence, the term is synchro-tuned. Disruptions in circadian rhythms can adversely affect various functions such as sleep, alertness, cognition, mental acuity, motor skills, and metabolism. The importance of maintaining a balanced circadian rhythm is increasingly emphasized for neurodegenerative disorders.

Circadian neurons work together to build a fine-grained regulatory system by forming a complex neural network that interconnects with other biological clock-related nuclei^[19]. These neurons regulate circadian rhythms in organisms by releasing neurotransmitters and hormones that influence the function of downstream target cells. The circadian system consists of interconnected clock oscillators in the suprachiasmatic nuclei (SCN) of the hypothalamus and certain active peripheral organs in terms of metabolism. It regulates daily behavioral and physiological rhythms encompassing sleep/wake cycles, fasting/feeding patterns, metabolic activities, body temperature, and endocrine functions. This system significantly influences daily feeding habits^[20], sleep-wake patterns regulation^[21], neuroendocrine hormonal secretion^[22], and maintaining homeostasis and metabolic processes, including the autonomic control of blood pressure and body temperature. In its intrinsic state, the circadian rhythm follows a natural period known as tau, slightly exceeding the length of the 24-h light-dark cycle (24.18 h) observed in humans^[23].

Relative genes of circadian rhythms

The hypothalamic SCN, acting as central circadian pacemakers in mammals, communicates synchronized signals to various circadian oscillators within the brain, tissues, and organs. Specialized cells containing intrinsic photosensitive melanopsin (MOP) receive light signals necessary for circadian regulation. Melanopsin is found in a small subset of retinal ganglion cells, which project toward the SCN through the retino-hypothalamic bundle^[24,25]. Neuropeptides such as vasoactive intestinal peptide (VIP), arginine vasopressin (AVP), and gastrin-releasing peptide (GRP) primarily synchronize oscillatory neurons within the SCN^[26-31]. Peripheral tissues’ rhythmicity is predominantly coordinated by glucocorticoid signaling within the SCN^[32-34]. While the hypothalamic SCN serves as the primary circadian pacemaker, most peripheral organs and tissues can exhibit circadian oscillations independently despite their interactions with each other and the system as a whole^[35]. At a molecular level, the generation of circadian oscillations involves a complex network of genes referred to as “clock genes,” which include the clock circadian regulator (CLOCK), aryl hydrocarbon receptor nuclear translocator-like (ARNTL, also known as BMAL1), period circadian regulators 1 (PER1) and 2 (PER2), and cryptochrome circadian regulators 1 (CRY1) and 2 (CRY2). These encoded proteins have critical functions in regulating circadian rhythmicity.

The core oscillator exhibits high consistency in function and compositional principles across eukaryotes. In Drosophila, the positive edges of the clock are formed by CLOCK and CYCLE, which bind to E-box-containing enhancers upstream of the period (per) and timeless (tim) genes to induce their transcription^[36-38]. When they reach critical levels in the cytoplasm, PER and TIM interact physically, translocating into the nucleus where they inhibit CLOCK-CYCLE function, thus repressing their own transcription^[39,40]. TIM undergoes photodegradation throughout the morning via the cytochrome (CRY)-dependent pathway^[41]. When TIM is experiencing a loss, the direct homolog of mammalian casein kinase 1ε (CSNK1E) destabilizes PER, leading to its ubiquitination and subsequent proteasomal degradation^[42,43]. The simultaneous degradation of PER and TIM initiates a new circadian cycle. Independent peripheral circadian oscillators exist in some non-mammalian vertebrates and numerous invertebrates, such as zebrafish and Drosophila^[44,45]. Multi-oscillatory systems, found in non-mammalian vertebrates like birds and lizards^[46], involve direct responses to environmental cues by the retina and pineal gland.

Molecular clock in transcriptional autoregulatory feedback loop begins with the activation of BMAL1 and CLOCK, prompting the expression level of PER and CRY genes at the beginning of a cycle. In a negative feedback mechanism, the PER/CRY repressor complex translocates into the nucleus to inhibit BMAL1/CLOCK activity^[47]. This loop operates with a genetically predetermined period lasting approximately 24 h and is primarily entrained by environmental light cues. Additionally, CLOCK-BMAL1 controls a myriad of target genes downstream, referred to as clock-regulated genes. The circadian system, an intricately regulated network, involves the oversight of PER and CRY protein stability by E3 ubiquitin ligase complexes known as S-phase kinase-associated protein 1 (SKP1)-cullin (CUL1)-F-box protein. Furthermore, casein kinase 1ε/δ (CSNK1E/D) and AMP kinase (AMPK) influenced the phosphorylation of PER and CRY proteins. Moreover, the respective E3 ubiquitin ligase complexes and the 26S proteasome complex, respectively, led to their subsequent polyubiquitination and degradation.

Aging and circadian rhythm

Circadian rhythms in mammals govern daily (approximately 24-h) oscillations in behavior and physiology, harmonizing internal processes with environmental timing cues to ensure optimal adaptation. Recognized is the attribution of circadian dysfunction in older adults, in part, to the SCN’s degradation, recognized as the mammals’ core circadian clock. The central clock is located in the hypothalamus’ SCN, orchestrating the synchronization of peripheral clocks in body tissues through both humoral and neural signals. These peripheral clocks operate autonomously at the cellular level, are influenced by the central clock, and do not necessitate SCN inputs to generate rhythmic patterns. The endocrine system also demonstrates circadian regulation. During aging, circadian rhythms show reduced amplitude and phase shifts, which may impact the organism’s ability to adapt to environmental changes and potentially affect tissue homeostasis, sleep regulation, behavior, health status, and lifespan^[48].

The aging process is closely linked to changes in circadian rhythm, which can accelerate aging^[49]. There is an increasing indication of the interplay between changes in circadian rhythmicity and aging naturally. Chronic conditions such as obesity, diabetes, cancer, cardiovascular disease, and neurodegenerative disorders are significantly influenced by aging. In aged mice, epidermal and muscle stem cells continue to exhibit circadian rhythmicity at a cellular level. However, the oscillatory transcriptome of aged muscle stem cells undergoes reprogramming^[50], shifting the focus of gene expression toward stress responses rather than maintaining homeostasis. Likewise, aging alters the circadian function of the liver in mice, resulting in a notable decrease in protein acetylation oscillations^[51]. Calorie restriction has been shown to reverse the aging-related reprogramming in both scenarios, hinting at potential benefits. Additionally, therapeutic interventions such as oxidized nicotinamide adenine dinucleotide (NAD⁺) precursors can modulate the circadian effects of aging by deacetylating Lys680 on PER2, thereby enhancing the binding of BMAL1 to chromatin across the genome^[52]. This observation suggests that it is feasible to intervene in the circadian output tied to natural aging through methods such as calorie restriction and specific treatments.

Epigenetic modification of circadian rhythms

Core clock genes are not the sole factor in modulating circadian transcription. It is also important to notice the genetic and epigenetic factors. Therefore, the feedback loop of the core circadian loop fails to explain all results solely, particularly those related to human behavioral traits and disorders^[53,54]. The key mechanism in epigenetic factors is translating environmental influences into the expression level of the circadian rhythm gene. Throughout the day-night cycle, dynamic shifts in the epigenetic landscape occur^[55]. Gene expression oscillations are collectively controlled by the collaboration between circadian transcription and chromatin modifications of circadian rhythms. Promoter regions of circadian clock genes (CCGs) exhibit rhythmic histone acetylation (AcH3K9 and AcH3K14)^[56], connecting the histone acetyltransferase (HAT) p300 (EP300) with CLOCK’s intrinsic HAT activity^[56,57]. Nonetheless, the NAD⁺-dependent deacetylase sirtuin 1 (SIRT1) counterbalances this activity^[58,59], dynamically regulating the circadian acetylation of histone and non-histone proteins.

CDC-like kinases (CLKs) utilize histone acetylation/deacetylation as a crucial epigenetic mechanism to regulate circadian rhythms. The regulation of histone H3 acetylation at the CRY and PER promoters is controlled by the EP300 in cooperation with the CLOCK/BMAL1 complex, impacting gene expression^[60]. HAT activity is also exhibited by CLK within the CLK-BMAL1 complex, leading to the recruitment of CRY1 by acetylated BMAL1, resulting in transcriptional repression^[56,61]. During the transcriptionally active phase, there is a peak in histone H3 acetylation levels in PER1, PER2, and CRY1 promoters^[62]. Circadian rhythms, addiction-related behaviors, memory formation, and metabolism are significantly influenced by histone deacetylases (HDACs)^[57,62]. The recruitment of HDAC3 by nuclear receptor corepressor 1 (NCOR1) represses the expression level of BMAL1, thereby influencing circadian rhythms. Fluctuations in recruiting HDAC3, along with nuclear receptor subfamily 1 group D member 1 and NCOR1, lead to the formation of an HDAC3/NR1D1/NCOR1 complex that correlates with the oscillatory transcription of numerous genes^[63,64]. Treatment with HDAC inhibitors results in increased acetylation of H3 and impacts the expression of PER2. SIRT1, a NAD⁺-dependent HDAC, binds to CLK-BMAL1 to directly interact with clock genes, promoting the deacetylation and degeneration level of PER2. This connection links the metabolic state to the circadian system. Besides its role in circadian regulation, SIRT1 is also involved in various brain functions such as aging, neurodegeneration, synaptic plasticity, and memory formation.

The regulation level of circadian rhythm gene expression is significantly influenced by histone methylation and demethylation processes^[65-67]. Various histone methyltransferases and demethylases participate in the rhythmic methylation of Histone H3 Lysine 4, 9, 27, 36 (H3K4, H3K9, H3K27 and H3K27)^[66,67]. The suppressor of variegation 3-9 homolog 1 (SUV39H1) is key in the rhythmic di-methylation of H3K9 (H3K9me2) and is responsible for recruiting CLOCK-BMAL1 to the E-box regions of clock-controlled gene promoters [Figure 2]^[65,68]. The interaction between SUV39H and PER2 could potentially regulate the formation of rhythmic heterochromatin during the repressive phase through H3K9me2 and chromobox 5 (CBX5) binding at D-box binding PAR bZIP transcription factor (DBP), PER1, and PER2^[54,66]. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) methyltransferase contributes to histone methylation, including di- and tri-methylation of H3K27 (H3K27me2 and H3K27me3), influencing the circadian gene expression of PER1 and PER2. The lysine demethylase 1A (KDM1A), lysine demethylase 5A (KDM5A), and ARID domain-containing histone lysine demethylase 1a (JARID1A) are crucial partners of CLOCK-BMAL1, enhancing its transcriptional activity. KDM1A removes methyl groups from H3K4 and H3K9, interacting with CLOCK and BMAL1, playing a pivotal role in the circadian clock mechanism and the regulation of clock-controlled gene expression^[69]. In contrast, JARID1A interacts directly with CLOCK-BMAL1 and is involved in regulating circadian gene expression, with additional functions impacting HDAC1 recruitment without altering H3K4me3 methylation. Furthermore, the histone demethylase, lysine demethylase 8 (KDM8)^[70], demethylates H3K36 and is responsible for recruiting various modifications, playing a role in modulating heterochromatin on a circadian timescale.

Figure 2. The core components of the circadian oscillator within the cell autonomously regulate the 24-h gene expression cycle, and a series of pathological alterations are caused by the loss of BMAL1. The 24-h rhythm of gene expression and the absence of BMAL1 are governed by the cell-autonomous core components of the circadian oscillator. Within the primary loop, as well as the core loop, the heterodimerization of CLOCK and BMAL1 drives the expression of PER and CRY genes in the morning, mediated by E-box elements. Subsequently, the PER and CRY proteins constitute a repressor complex that inhibits the transactivation of CLOCK and BMAL1. In the secondary loop, REV-ERBα, REV-ERBβ, and RORα, RORβ, RORγ subfamilies of nuclear receptors act as repressors and activators to antagonistically regulate BMAL1 and other target genes at RORE promoter elements at nighttime. The D-box, activated by PAR-bZIP proteins like DBP and repressed by E4BP4, plays a crucial role in the transcriptional regulation of circadian clock-controlled genes. Various ligands and small molecules have been identified to influence core clock components and circadian functions. Loss of BMAL1 is associated with neurodegeneration in AD and other age-related dementias. The absence of BMAL1 in the brain adversely affects astrocytes, microglia, neurons, and pericytes, resulting in increased oxidative stress and inflammation, as well as alterations in neurotransmitter signaling. These changes may contribute to astrogliosis and astrocyte activation, impaired microglial phagocytosis, and neuronal injury, which, in turn, lead to synaptic damage, alterations in Aβ dynamics, and loss of brain connectivity - key pathological features of AD and related dementias. Potential signaling pathways involved include GST-mediated protein glutathionylation, the SIRT1-BMAL1 axis, GABA signaling, NAD⁺ depletion-related cyclic GMP-AMP synthase, and interferon gene stimulating factor activation, all of which play potential roles in AD. BMAL1: Aryl hydrocarbon receptor nuclear translocator-like; CLOCK: clock circadian regulator; PER: period circadian regulators; CRY: cryptochrome circadian regulators; DBP: D-box binding PAR bZIP transcription factor; AD: Alzheimer’s disease; Aβ: amyloid-β; GST: glutathione S-transferase; GABA: gamma-aminobutyric acid; NAD⁺: nicotinamide adenine dinucleotide; GMP-AMP: guanosine monophosphate - adenosine monophosphate.

AD and related dementias

The relationship between sleep and disease-specific pathways, particularly regarding Aβ, a pathogenic protein associated with AD, has been robustly documented. Aβ, known to form amyloid plaques, aggregates in the brain of patients with AD years before cognitive impairment occurs, serving as an early AD biomarker^[72]. Aβ concentrations fluctuate in the brain’s extracellular space following a circadian rhythm, with an increase during waking periods and a decrease during rest, irrespective of light cycles. These oscillations in Aβ are closely tied to changes in neuronal metabolic activity during sleep and wakefulness^[73]. Preclinical research using transgenic mouse models with Aβ deposition has shown that sleep deprivation accelerates Aβ plaque formation, whereas sleep promotion through orexin antagonist drugs inhibits Aβ plaque deposition. Genetic deletion of the hypocretin neuropeptide precursor (HCRT) gene, which modulates wakefulness and regulates feeding and metabolism, results in modest increases in sleep duration and effectively suppresses Aβ plaque formation in AD mouse models^[74]. Aβ deposition in the brains of transgenic mice gradually increased with age, especially peaking in old age^[73]. This result suggests that age is an important factor in Aβ deposition and is closely related to the risk of developing neurodegenerative diseases, such as AD.

Epidemiological studies have corroborated these findings, identifying fragmented or deficient sleep as a risk factor for future symptomatic AD in cognitively normal individuals^[75]. Moreover, individuals with Aβ plaque pathology experience noticeable declines in sleep quality prior to the onset of cognitive symptoms. Studies in humans have also shown a correlation between the levels of orexin and tau protein, another hallmark of AD, in cerebrospinal fluid (CSF). Higher levels of orexin-A, promoting wakefulness, are linked with increased levels of phosphorylated tau, a well-established marker of neurodegeneration in AD, in the CSF^[76]. However, the relationship between sleep and tau pathology is yet to be fully elucidated. In summary, sleep deprivation contributes to Aβ plaque formation, while orexin modulation and improved sleep quality show promise in inhibiting Aβ plaque deposition. Further research is needed to obtain a deeper comprehension of the intricate relationship between sleep, Aβ, tau, and neurodegeneration in AD.

Patients with moderate-to-severe AD experience more pronounced disruptions in their circadian rhythms than healthy adults of a similar age. These disruptions manifest as increased sleep fragmentation, reduced amplitude, and phase delay rather than the typical advanced circadian phase seen in healthy aging^[77]. The phase delay of temperature and hormone rhythms in AD may partly contribute to the phenomenon of “sundowning”, characterized by heightened behavioral and neuropsychiatric symptoms in patients with AD around sunset^[78]. Commonly, sleep-wake disorder observed in AD patients is irregular sleep-wake rhythm disorder (ISWRD), differing from the advanced sleep-wake rhythm disorder commonly seen in older adults without cognitive impairment^[79]. In cases of severe AD, the lack of a distinct 24-h sleep-wake cycle, manifested by prolonged periods of wakefulness at night and erratic episodes of sleep throughout the day, serves to define ISWRD.

In recent times, there has been a notable increase in the number of studies examining patients with varying degrees of cognitive impairment. These studies have identified discrepancies in their circadian patterns compared to previous research that predominantly focused on patients with moderate to severe AD^[77]. This discrepancy may be attributed to the disparate types or degrees of cognitive impairment documented in these more recent studies. The included studies encompassed patients with preclinical AD^[80], mild cognitive impairment^[81], mild AD^[82,83], moderate to severe AD^[83], global AD^[84], and early onset dementia^[85]. All these studies reported disruptions in rest-activity rhythms and sleep timing as behavioral markers of circadian rhythm disruption. Overall, these studies found that rest-activity rhythm fragmentation was high^[80,85]; however, a modest decrease or absence of alteration was observed in the amplitude of rest-activity or melatonin rhythms^{[80,82,84-86]}. Therefore, there is a need to focus on patients with different severities of AD.

PD

PD, a progressive neurodegenerative movement disorder, affects 2%-3% of individuals aged over 65 globally. The primary diagnostic markers of PD are motor function abnormalities, primarily caused by a reduction in dopamine levels within the basal ganglia system. Moreover, circadian rhythms also exert influence over inflammation and oxidative stress, both of which are pivotal pathologies linked with PD-specific neurodegeneration. More critically, neurodegeneration and pathological protein aggregation resulting from various etiologies can, conversely, cause sleep disturbance and disruptions of circadian rhythms, thereby establishing a deleterious feedback loop.

PD exhibits disruptions in the typical 24-h rhythms of motor and non-motor symptoms. Unlike those with AD, individuals with PD and circadian rhythm disturbances experience a decrease in the amplitude of their circadian rhythm, albeit lacking a statistically significant shift in circadian phases^[87-92]. Indeed, there is evidence to suggest that fluctuations in the non-motor symptoms of PD do occur on a seasonal basis. These findings indicate that individuals with PD experience a greater burden of symptoms during the winter months than during the summer months^[93]. Some have proposed that these seasonal fluctuations in PD symptoms may be attributed to changes in circadian regulation. It is well-established that dopamine metabolism is closely intertwined with circadian homeostasis. In a male Drosophila model, disruption of circadian rhythms exacerbated the degeneration of dopaminergic neurons within them^[94].

Furthermore, circadian rhythms of microglia have a potential role in PD and other neurodegenerative diseases^[95]. A substantial body of evidence from numerous studies indicates that the circadian clock in microglia may regulate several processes, including the release of inflammatory cytokines, autophagy, phagocytosis, and redox homeostasis, under physiological conditions. However, some theories require further investigation^[96,97]. Consistently, disturbances in circadian rhythms or sleep disorders may exacerbate PD progression through mechanisms involving microglia-mediated neuroinflammation, oxidative stress, and protein homeostasis^[98,99].

Disruptions to the sleep-wake rhythm are extensively documented symptoms in neurodegenerative diseases such as PD. Sleep-wake disturbances, impacting up to 80% of patients with PD^[100], represent the most common non-motor symptom in PD. Various studies have reported excessive daytime sleepiness (EDS) and changes in sleep timing among patients with PD^[89,101-103], indicating that they are more than twice as likely to experience EDS than healthy older adults^[87,88]. To date, limited evidence suggests a slightly delayed sleep onset in patients with PD compared to healthy controls^[89], with some studies not finding significant differences^[90,92]. An Australian study revealed a notable decrease in the mesor and amplitude of the core body temperature rhythm in patients with PD compared to age-matched controls^[90]. Research on melatonin secretion rhythms in patients with PD using different samples, such as plasma, serum, and saliva, indicates lower circulating levels of melatonin in patients with PD than their healthy counterparts, although the timing of melatonin onset remains similar^[87,89,92]. Additionally, patients with PD may lack the typical nocturnal decline in blood pressure, leading to an increased risk of cardiovascular issues, including nocturnal hypertension^[91]. For example, one study conducted in Spain found that 71.1% of patients with PD did not demonstrate the expected decrease in blood pressure throughout a 24-h period, as recorded by ambulatory blood pressure monitoring^[91].

ALS

Sleep-related breathing disorders represent the most prevalent sleep disturbance among patients with ALS, with a substantial body of documented evidence^[104]. Modifications in respiration during the sleep cycle may foreshadow the onset of respiratory symptoms upon waking, even in patients with unimpaired respiratory function. However, as ALS progresses, the weakening of respiratory and upper airway muscles and the diaphragm precipitates nocturnal hypoxia and hypoventilation^[105,106]. Sleep disorders in ALS encompass reduced sleep quality and EDS, with the latter being more common and linked to disease severity and cognitive decline^[102]. Insomnia, featured by difficulties with initiating or maintaining sleep or with waking up at an early hour, is frequently observed in patients with ALS^[104,107]. Nonetheless, sleep disorders that are not associated with respiratory issues have been less extensively investigated in the context of ALS.

HD

HD is a progressive neurodegenerative disorder that is typified by a range of symptoms, including motor deficits, cognitive decline, and psychiatric manifestations. Despite the predominance of sleep disturbances in HD, they have not been as thoroughly investigated as in other neurodegenerative conditions^[108]. Recent findings suggest the presence of circadian dysregulation in HD, with flattened melatonin secretion rhythms and delayed circadian phases observed in HD patients^[109]. Circadian disruption has been observed in animal models of HD, exemplified by transgenic R6/2 mice showing increased daytime activity and decreased nighttime activity^[110]. Large animal models of HD, like transgenic sheep expressing the mutant Huntingtin protein, display significant circadian abnormalities, including behaviors resembling human sundowning^[111]. The observed behavioral changes coincide with abnormal expression of the clock genes BMAL1 and PER2 in the striatum, motor cortex, and SCN.

While the observed circadian abnormalities provide a basis for considering circadian-based interventions for individuals with HD, systematic investigations of such interventions in patients with HD are lacking. Limited studies examining the effects of exercise on sleep in HD have not consistently demonstrated substantial improvements in sleep metrics^[112]. Pharmacological interventions have shown efficacy in restoring circadian rhythms in R6/2 mice^[113]. Other zeitgebers, besides light, such as feeding schedules, have demonstrated positive impacts on rest-activity cycles in the HD model of R6/2 mice. These effects are likely mediated through the activation of the food-entrainable oscillator, which operates independently of the SCN^[114]. Exploring sleep disturbances in HD may further help to inform future therapeutic strategies to slow disease progression^[115]. A recent study showed promise in restoring locomotor function and normalizing disrupted circadian gene expression in the HD model of Drosophila using melatonin and curcumin^[116], a polyphenol derived from turmeric known to activate BMAL1 and potentially modulate circadian clock processes^[116].

Underlying mechanisms between circadian rhythms and neurodegenerative diseases

The impact of neurodegenerative pathology on circadian function varies across different diseases. In AD, research has demonstrated a reduction in crucial neuronal populations in the SCN, specifically those that express AVP or VIP^[84]. The reduction in the number of neurons expressing VIP in the SCN is associated with the circadian dysfunction observed before death. However, the precise mechanisms underlying this neuronal loss remain uncertain. Unlike the regions heavily affected by Aβ plaque or neurofibrillary tangles, the SCN does not show significant pathology. Transgenic mouse models of AD expressing mutant human APP, tau, or both also exhibit circadian abnormalities, but establishing a direct link with pathology has proven challenging, hindering mechanistic understanding^[117-119]. The Aβ peptide has been proposed to contribute to circadian dysfunction by promoting the degeneration of BMAL1 in cultured cells^[120,121]. Nevertheless, at present, there is no indication to imply that Aβ directly interacts with the circadian clock in animals or humans. Disrupted circadian rhythms, attributed to dysregulated methylation of the BMAL1 promoter, have been observed in fibroblasts from patients with AD and post-mortem brain samples, indicating an epigenetic mechanism underlying the disruptions of circadian rhythms in AD.

Conversely, potential mechanisms through which circadian rhythms impact neurodegenerative diseases have been postulated^[122]. Disruption of circadian rhythms may influence the advancement of neurodegenerative diseases by changing sleep duration, resulting in disjointed nighttime sleep and increased daytime napping. Sleep deprivation can potentially modify Aβ dynamics in humans and exacerbate Aβ and tau pathology in mouse models^[123-125]. Furthermore, it can escalate levels of inflammatory and neuron-specific injury biomarkers in the human CSF^[126]. Moreover, sleep deprivation has been implicated in triggering protein clearance from the brain in mouse models, promoting inflammation, and impacting synaptic homeostasis associated with neurodegenerative diseases by modulating immune responses^[127,128]. Interventions aimed at enhancing sleep quality are generally intended to counteract the consequences of circadian rhythm disturbances. Nonetheless, research has revealed that mutations in clock genes can provoke neuropathology resembling astrocyte proliferation in mice, even without changes in sleep patterns. This observation suggests that the impacts of circadian terminals on the brain cannot be solely attributed to alterations in sleep.

Circadian dysfunction may impact neurodegeneration through the regulation of immune responses. The timing of exposure to inflammogens significantly influenced the peripheral immune response in mice. Circadian rhythms strongly affect the level of inflammation^[129,130]. In a mouse model of experimental autoimmune encephalitis, disease severity at later stages was notably influenced by the timing of immunization. Rodents with Bmal1 deleted in myeloid cells experienced exacerbated pathology^[131,132]. In addition, global and brain-specific deletion of Bmal1 in neurons leads to cell-autonomous dopaminergic neurodegeneration^[133]. Microglia and astrocytes, the key innate immune cells in the brain, have functional circadian clocks that control inflammatory responses^[96,134]. Disruption of the circadian clock function in the mouse brain by deleting Bmal1 led to widespread activation of astrocytes and synaptic degeneration. These findings underscore the pivotal function of the core clock function in preserving immune equilibrium within the brain^[135]. Disturbances in circadian rhythms induced by non-24-h light-dark cycles have been demonstrated to elevate the activation level of glial and inflammation levels of neurons, exacerbating disease progression in mouse models of ALS and PD^[136,137]. Therefore, circadian dysfunction is likely involved in certain aspects of neuroinflammation that can impact neurodegeneration in distinct disease states.

The circadian clock’s influence on protein homeostasis and quality control may affect protein deposits in neurodegenerative diseases. Diurnal variations in Aβ levels have been observed in human CSF and mouse interstitial fluid^[73,120,138]. Furthermore, disrupting the circadian clock in a mouse model of β-amyloidosis and AD accelerated the accumulation of amyloid plaques^[138]. In a broader context, regulating circadian rhythms within protein quality control mechanisms, such as autophagy, may also lead to the circadian influence on protein aggregation^[139,140]. The glymphatic system, a perivascular fluid clearance system mediated by glial cells, eliminates aggregated proteins from the brain in bulk. While this process is associated with sleep, its relationship with the circadian system and the involvement of glial clocks remains unclear^[127]. Therefore, various identified and unknown mechanisms could link the circadian clock to neurodegenerative conditions.

RNA modifications

Chemical alterations to the bases or ribose sugar in RNA molecules are known as RNA modifications. Over 150 different changes have been found to date^[145]. These modifications exert a pivotal influence on the nervous system, regulating cellular heterogeneity and molecular complexity through the modulation of gene expression. The chemical modifications of RNA bases and sugar residues constitute the neuronal epitranscriptome. These RNA modifications are regulators of RNA stability, transport, and translation. They affect diverse downstream signaling pathways. Specific chemical modifications of biological components are an effective way to regulate molecular activity. Several pathogenic diseases, including cancer, cardiovascular abnormalities, and neurodegenerative disorders, can result from perturbations in the external transcriptome.

Commonly observed RNA modifications can contribute to various neurodevelopmental and neurodegenerative disorders, including methylation and isomerization of adenine and cytosine bases. Representative post-transcriptional processing steps for eukaryotic RNA include 5’ capping, intron excision or splicing, and the insertion of a 3’ polyadenylated tail. Another significant pathway of post-transcriptional RNA modification involves the chemical alteration of RNA bases and sugar residues on the RNA backbone.

The functional impact of the epitranscriptome is evident and pervasive, manifesting in nearly all tissues. It especially contributes to the complexity of the nervous system by modulating various levels of RNA metabolism. Recent studies have emphasized the contribution of dysregulated RNA transport, splicing, stabilization, translation, or microRNA (miRNA) biogenesis to neurodegenerative diseases. RNA metabolism is crucial for maintaining proper brain function and consolidating learning-based memory in the adult brain.

While 170 distinct RNA modifications have been detected so far, only a few have been well characterized and particularly associated with neurologic disorders. Three groups of protein factors with substrate-specific enzymatic activities, termed writers, readers, and erasers, collectively edit epitranscriptome. They are collectively called RNA-modifying proteins (RMPs) and are responsible for depositing, reading, and removing RNA modification marks^[145]. Readers, which frequently alter RNA metabolism, recognize specific RNA modification marks, binding to these sites and activating downstream pathways. Mutations and variations in RMP abundance are associated with several diseases, including infertility, obesity, neurodegenerative and neurodevelopmental disorders, and cancer.

RNA metabolic mechanisms modulate the expression of proteins involved in developing, structuring, and functioning the brain over time. RNA-binding proteins (RBPs) adjust RNA metabolism, leading to complex gene expression patterns in different parts of the neuron. Other RBPs may counteract the functions of expression-mediated RBPs to balance RNA metabolism and gene expression in neurons throughout the brain. Maladjusted functioning and enrichment of RBPs can seriously disrupt control mechanisms, which can lead to neurodevelopmental or neurological diseases.

Quite many RNA modifications have been confirmed on coding and non-coding RNAs (ncRNAs), with only a limited number extensively studied or linked to diseases. Among the well-studied RNA modifications are m⁶A, m¹A, m⁵C, pseudouridine, and RNA editing [Table 3].

RNA modifications and neurodegenerative diseases

RNA modifications and circadian rhythm

Linkage among RNA modifications, circadian rhythms, and neurodegenerative diseases

Studies have demonstrated that RNA modification affects the development and progression of degenerative neurological diseases. In the post-transcriptional modification of mRNA, A-to-I RNA editing, m⁶A RNA methylation, and AS assume pivotal roles across all stages of the neuronal cell life cycle, with alterations in their mechanisms significantly contributing to aging and neurodegeneration^[203]. In the AD brain, neurons show decreased m⁶A levels and expression of m⁶A METTL3, and METTL3 knockdown leads to decreased m⁶A modification of neurons in the hippocampus, leading to significant memory deficits, suggesting that METTL3-mediated m⁶A dysregulation may lead to neurodegeneration in AD^[204]. The overexpression of METTL3 rescues synaptic damage and cognitive impairment in vivo induced by Aβ^[204]. Inactivation of FTO has been demonstrated to alter the delivery of neuronal dopamine^[205], implicating adenosine RNA methylation in regulating dopaminergic signaling pathways and suggesting its potential involvement in the etiology of PD. These studies suggest a link between RNA methylation and neurodegenerative diseases.

The co-occurrence of TDP-43 aggregation and decreased expression level of ADAR2 in the same motor neurons is a characteristic of sporadic ALS^[206]. ADAR2 primarily targets the Q/R site in glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) pre-mRNA. The absence of ADAR2 leads to unedited GRIA2, resulting in heightened intracellular calcium influx and consequent slow motor neuron demise^[207]. ADAR2 deficiency triggers calpain activation, initiating the progressive formation of TDP-43 aggregates and subsequent disease progression^[206]. In patients with AD, GRIA2-Q/R editing was less efficient at 30 different sites in the brain^[182]. The decrease in ADAR2 levels was associated with diminished Q/R site editing, potentially contributing to the initiation or progression of ALS. Within the realm of neurodegenerative diseases, several have been predominantly associated with alterations in AS, including AD, PD, ALS, FTD, and familial dysautonomia (FD).

A comprehensive transcriptomic analysis conducted on the dorsolateral prefrontal cortex of 450 subjects from two distinct aging cohorts revealed the presence of 84 genes exhibiting aberrant pre-mRNA splicing events, which were found to be significantly associated with AD^[208]. In PD, the 112-amino-acid SNCA transcript is generated by the removal of exon 5, resulting in the absence of S129, a pivotal phosphorylation site crucial for SNCA clearance, aggregation, and toxicity^[209]. Removal of exon 3 leads to the production of the 126-amino-acid SNCA transcript, which is elevated in stages 3 and 4 in patients with PD^[210]. Isoforms lacking exons 3-5 or 2-7 are upregulated in PD, and AS variants of parkin RBR E3 ubiquitin protein ligase (PRKN) lacking exon 4 are upregulated in sporadic PD^[211]. Mutations in elongator acetyltransferase complex subunit 1 (ELP1) in FD disrupt the 5’ splice site within intron 20, causing exon 20 to be skipped, resulting in a frameshift that generates premature termination codons in exon 21, triggering nonsense-mediated decay of IKBKAP transcripts. The absence of these transcripts contributes to the progressive degeneration of autonomic and sensory neurons^[212]. Therefore, RNA modifications linked with circadian rhythms, including A-to-I RNA editing, m⁶A RNA methylation, and AS, are intricately linked to the pathogenesis of neurodegenerative diseases, influencing key processes in neuronal function and survival.

Methods

RNA-based therapeutics have become increasingly popular in treating various neurological diseases, offering diverse potential applications. Various therapeutic approaches, including RNA editing, antisense oligonucleotides (AONs), small interfering RNAs (siRNAs), RNA aptamers, RNA-based vaccines, and mRNA drugs, are proving effective in treating diverse conditions. Among these modalities, various studies have extensively researched the design, chemistry, and utilization of AONs^[213-215]. Despite advancements in rare genetic diseases and neurodegenerative disorders, overcoming the challenge of safely and effectively delivering these therapies to the brain remains a significant obstacle. Furthermore, the safety concerns associated with RNA-based therapies encompass not only challenges in delivery but also issues related to suboptimal pharmacological properties, cell-specific targeting, immune-related toxicities, and potential long-term adverse effects. The development of personalized RNA-based treatments for ultra-rare diseases is being explored alongside their application in more common conditions such as AD and stroke. The potential of RNA-based therapeutics in addressing behavioral disorders and CNS tumors has not been fully realized. However, with advancements in the field, their transformative impact on neurology is promising. There are three groups of RNA-based therapies classified based on different mechanisms: those that act on nucleic acids [such as AONs or via the RNA interference (RNAi) pathway], those targeting proteins (utilizing RNA aptamers), and mRNA drugs designed to produce proteins.

Currently, the predominant form of RNA-based therapeutics is AONs. AONs consist of DNA or RNA sequences, usually 15-20 bp in length, and are modified to enhance stability. The selection of specific chemicals relies on the intended application, with all clinically approved AONs belonging to either oligomer of phosphorodiamidate morpholino or 2’-O-methoxyethyl^[216]. Moreover, splice-switching AONs (ssAONs) obstruct intronic or exonic cis-regulatory elements to manipulate the target exon’s inclusion or exclusion. These ssAONs target splice sites, exonic splicing enhancers or silencers, and their respective intronic counterparts. Furthermore, ssAONs can be effective in inhibiting the use of cryptic splice sites or introducing new splice sites for donors or acceptors^[217].

RNAi, a regulatory mechanism in most eukaryotic cells for direct control of gene activity, has emerged as a mechanism for drug action in developing RNAi-based therapies [Figure 3]. Researchers have been familiar with RNAi for over twenty years, a natural process where short RNA strands, like siRNAs, induce specific gene suppression. siRNAs are double-stranded RNA (dsRNA) molecules that separate into single strands and selectively bind to target mRNA sequences. This binding initiates a series of events leading to the degradation and cutting of the target mRNA, halting translation and subsequent gene expression and function. siRNAs offer significant therapeutic potential by enabling the specific targeting and silencing of mRNAs from genes previously deemed challenging to target pharmacologically.

Figure 3. Overview of RNAi and AONs. (A) RNAi pathways (siRNAs on the left side; miRNAs on the right side). siRNAs are usually of exogenous origin, while miRNAs are typically derived from transcripts possessing internal hairpins. Following Dicer-mediated processing, the RNA is recruited into AGO2 to constitute the RISC. siRNAs target mRNAs with quite precise sequence complementarity, resulting in mRNA cleavage by AGO2. In contrast, miRNAs recognize target sites with imperfect complementarity, predominantly in the 3’UTR of mRNAs, resulting in translational repression, deadenylation, and destabilization of the target. Additionally, miRNAs with precise complementarity can induce endonucleolytic cleavage by AGO2. Drosha, an RNase III enzyme, cleaves miRNA precursors in the initial stages of miRNA production by processing pri-miRNAs into pre-miRNAs, which are further processed by Dicer to produce mature miRNAs. AGO proteins play a central role in the RISC complex, which combines siRNAs with AGO proteins and Dicer enzymes to achieve gene silencing through the cleavage of target mRNA by the antisense strand of siRNAs; (B) AONs pathways (ssAONs on the left side; Gapmer AONs on the right side). ssAONs are directed to the nucleus to bind to their target pre-mRNA, which disrupts the splicing process by blocking the spliceosome. The specific example highlighted involves an AON targeting exon 51 of the dystrophin gene, leading to exon skipping. On the other hand, Gapmer AONs, shown on the right side, can trigger RNase H-mediated cleavage of the target mRNA in both the nucleus and the cytoplasm. RNAi: RNA interference; AONs: antisense oligonucleotides; siRNAs: small interfering RNAs; miRNAs: microRNAs; AGO2: Argonaute protein 2; RISC: RNAi‐induced silencing complex; 3’UTR: 3’ untranslated region; ssAONs: splice-switching AONs.

Almost all human protein-coding genes exhibit AS, which involves processes such as exon inclusion or exclusion, switching between different splice sites, retaining introns, selecting mutually exclusive exons, and other complex patterns of splice site selection^[218]. The regulation of these splicing events relies on the spliceosome, a large molecular complex responsible for catalyzing splicing reactions^[219]. Spliceosome assembly necessitates a coordinated interplay among trans-acting factors, encompassing small nuclear ribonucleoproteins, such as U1 and U2, constituting snRNAs and highly related proteins, in conjunction with approximately 150 accessory proteins. Recent breakthroughs in cryo-electron microscopy have provided detailed insights into the structure and mechanics of spliceosomes, revealing their structural foundation with unprecedented clarity [Figure 4].

Figure 4. Overview of AS and RNA editing. (A) In pre-mRNA splicing, boundaries between intron and exon are recognized as the splice sites, and two consecutive transesterification reactions occur. Internal introns are eliminated from precursor mRNA transcripts through a two-step splicing process. The spliceosome, consisting of 5 snRNP complexes and over 150 accessory proteins, serves to recognize, juxtapose, and facilitate intron excision. As depicted in this figure, ATP-consuming RNA helicases (in humans), namely DDX46, DDX39B, DDX23, SNRNP200, EFTUD2, DHX16, DHX38, DHX8, and DHX15, orchestrate the dynamic process of spliceosome assembly and facilitate the exchanges of snRNPs and other factors. These helicases navigate through kinetic barriers to drive spliceosome activation, catalysis, and product release; (B) A-to-I RNA editing, illustrated on the left, plays a role in neurodegenerative diseases like ALS, AD, and PD, while the right side focuses on m⁶A RNA methylation and its impact on these conditions. A-to-I RNA editing is a post-transcriptional process that alters dsRNA structures through the deamination of specific adenosines (A) to inosines (I) by the ADAR enzyme family. This modification has an impact on both coding and non-coding transcripts. The schematic diagram represents the m⁶A RNA modification process and its involvement in neurodegenerative disorders like AD, PD, and ALS. The methylation of RNA m⁶A is carried out by the methyltransferase complex (writers), including the core subunit heterodimer METTL3/METTL14, along with cofactors like WTAP. Furthermore, erasers such as FTO and ALKBH5 can reversibly remove m⁶A RNA modifications. Specific readers like YTHDF1-3 recognize and bind to m⁶A-modified RNAs, influencing RNA processing, transportation, translation, and stability. AS: Alternative splicing; A-to-I: adenosine to inosine; ALS: amyotrophic lateral sclerosis; AD: Alzheimer’s disease; PD: Parkinson’s disease; m⁶A: N⁶-methyladenosine; dsRNA: double-stranded RNA; ADAR: adenosine deaminase; WTAP: WT1-associated protein; FTO: alpha-ketoglutarate dependent dioxygenase; ALKBH5: alkB homolog 5.

The CRISPR-Cas13 system can potentially modify RNA, allowing for programmable regulation of AS, A-to-I, and C-to-U RNA editing, and m⁶A modifications^[220]. Catalytically inactive Cas13b (dCas13b) is combined with the ADAR’s deaminase domain in humans, which naturally deaminates adenosine to inosine in dsRNA, with CRISPR RNA guiding the editing process by creating a dsRNA structure around the target adenosine^[221]. For specific C-U exchange, the cytidine deaminase from the adenine deaminase domain of ADAR2 has been engineered and fused with dCas13b to create programmable RNA editing in mammalian cells^[222]. Cas13-based tools have been developed for the m⁶A of mRNA, utilizing YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) and YTHDF2 fused to catalytically inactive Cas13 ortholog from Prevotella sp. (dPspCas13b) proteins to modulate mRNA transcripts within cells^[223]. Fusing dPspCas13b with the methyltransferase domain of m⁶A writer METTL3 offers the possibility of cytoplasmic adenine N⁶ methylation, while similar tools target m⁶A demethylation at specific sites^[224,225]. The potential for site-specific Cas13-mediate[d RNA editing to correct disease-causing mutations presents new therapeutic avenues for addressing neurodegenerative diseases resulting from abnormal RNA methylation or editing.

RNA therapeutics that are currently in development to effectively target neurodegenerative diseases are examined and summarized in Table 4. While not exhaustive, it is hoped that these promising drugs will provide readers with useful information.

Current concerns

In recent years, advancements in RNA design and delivery have facilitated the development of RNA-based medicines across diverse applications, including therapeutics, vaccines, and diagnostics. However, before exploring the potential of RNA-based therapies, it is imperative to elucidate the latent safety concerns associated with these approaches. These concerns encompass not only fundamental challenges in accurate delivery to the designated location but also issues related to poor pharmacological properties, cell-specificities, immune-related toxicities, and potential long-term adverse effects.

Oligonucleotide (OGN)-based therapeutics represent the emerging class of agents that utilize nucleic acids to treat various gene diseases. OGNs include AONs, siRNAs, aptamers, and miRNA inhibitors. Two of the bright and extensively utilized platforms in OGN gene therapies are AONs and siRNAs, which both frequently target similar molecular entities. Given two clinical safety reports of drug-related severe thrombocytopenia associated with AONs and exacerbation of peripheral neuropathy with the siRNA revusiran, we must take steps to emphasize the safety of these OGN-based therapies^[234]. More specifically, it needs to be considered whether OGN-based drug toxicity is (i) only present in AONs vs. siRNAs, (ii) determined by platform specificity, (iii) caused by certain modifications or sequences independent of class, and (iv) based only on relative distinctions in potency while allowing for the use of the lowest therapeutically-effective dose.

While mRNA therapies have demonstrated advantages in safety, programmability, flexibility, and cost-effectiveness in design and production, several inherent properties of mRNA and technological limitations pose significant challenges. The primary issues include (i) instability (they are single-stranded, negatively charged, and degrade rapidly, necessitating strict cold-chain operation), (ii) innate immunity (stimulation of innate and adaptive immune responses and low immunogenicity are required for protein-replacement therapies), (iii) delivery (highly efficient and cell/tissue-specific delivery).

In the past few years, significant advancements have been achieved in utilizing RNA aptamers for therapeutic and diagnostic applications. Notably, selection strategies have been refined, and post-modification techniques to enhance the biostability of RNA aptamers have been developed. However, as nucleic acid biopolymers, the therapeutic potency in vivo of RNA aptamers is critically limited by their inherent physicochemical traits. These properties affect the pharmacokinetic properties, thus exposing the aptamers to challenges such as the susceptibility of unmodified aptamers to degradation by nucleases, the susceptibility of small aptamers to rapid excretion by renal filtration, and potential physiological toxicities such as polyanionic effects, unintentional tissue accumulation, dense accumulation of chemical modifications or conjugates, and nonspecific immune activation^[235-237].

Advancing technology in RNA offers a creative approach to developing novel therapeutics targeting those rare diseases and other challenging-to-cure ones. As RNA-based therapeutics have evolved, their challenges have become increasingly prominent, and in addition to focusing on the most notable delivery issues, potential side effects, cytotoxicity, and pharmacological properties cannot be ignored.

Promising potential

The use of ssAONs is attracting attention as potential therapeutics for dementia-associated diseases, as the role of RNA metabolism deficiencies becomes increasingly evident^[238]. Targeting RNA directly at the source of pathogenesis holds promise for more effective outcomes than downstream interventions through alternative therapies. Due to the crucial involvement of APP proteolytic processing in neurodegenerative processes, ssAONs have been investigated for their impact on APP regulation. Notably, ssAONs have been developed to trigger APP exon 17 skipping, which contains the cleavage site of γ-secretase essential for Aβ production, showing effectiveness both in vitro and in vivo^[239,240]. Additionally, ssAONs targeting tau, another significant factor in neurodegeneration, are also in development. Tauopathies manifest as the aberrant accumulation of the MAPT encoded by the MAPT gene.

RNA editing offers a multitude of appealing therapeutic possibilities. Neurological and neurodegenerative disorders are underpinned by A-to-I RNA editing. The dysregulation of RNA editing in AMPA receptors leads to epilepsy and related conditions. Several in vivo proof-of-concept studies have demonstrated the potential of ADAR-mediated RNA editing to rectify missense and nonsense mutations in support of therapeutic RNA editing. ADAR naturally participates in splicing regulation^[241], and the editing of splice sites in the genome can influence splicing^[242-244], bolstering the argument for therapeutic targeting of splice sites.

In conclusion, RNA modifications, encompassing various molecular techniques such as RNA editing, RNA aptamers, RNA biosensors, and in vitro transcribed mRNA therapeutics, hold significant promise as a therapeutic approach for a wide array of challenging neurodegenerative diseases. They have become a vital component of the molecular toolkit in biotechnology.