The influence of circadian rhythms and aerobic glycolysis in autism spectrum disorder

Intellectual abilities and their clinical presentations are extremely heterogeneous in autism spectrum disorder (ASD). The main causes of ASD remain unclear. ASD is frequently associated with sleep disorders. Biologic rhythms are complex systems interacting with the environment and controlling several physiological pathways, including brain development and behavioral processes. Recent findings have shown that the deregulation of the core clock neurodevelopmental signaling is correlated with ASD clinical presentation. One of the main pathways involved in developmental cognitive disorders is the canonical WNT/β-catenin pathway. Circadian clocks have a main role in some tissues by driving circadian expression of genes involved in physiologic and metabolic functions. In ASD, the increase of the canonical WNT/β-catenin pathway is enhancing by the dysregulation of circadian rhythms. ASD progression is associated with a major metabolic reprogramming, initiated by aberrant WNT/β-catenin pathway, the aerobic glycolysis. This review focuses on the interest of circadian rhythms dysregulation in metabolic reprogramming in ASD through the aberrant upregulation of the canonical WNT/β-catenin pathway.


Introduction
Intellectual abilities and their clinical presentations are extremely heterogeneous in autism spectrum disorder (ASD). ASD includes Asperger, autism and pervasive developmental disorder-not otherwise specified (PDD-NOS). ASD is characterized by numerous and complex etiologies, including inflammation, metabolic, environmental or genetic determinants. Nevertheless, the main causes of ASD remain unclear. ASD is diagnosed within the first 3 years of life. Social interaction impairment, repetitive and restrictive behaviors or stereotypic patterns of behaviors characterize the different symptoms of ASD 1 . Early diagnosis is major issue for better prognosis and therapeutic care 2,3 .
ASD is frequently associated with sleep disorders 4 . Brain structures and functions development are considered as dynamic processes, dependent on genetic and social/physical environmental cues, involving homeostasis processes at each time, and maturation of new functions in time. Biologic rhythms are complex systems interacting with the environment and controlling several physiological pathways, including brain development 5 and behavioral processes 6 .
Recent studies have shown that the deregulation of the core neurodevelopmental signaling is correlated with ASD clinical presentation. One of the main pathways implicated in developmental cognitive disorders is the canonical WNT/β-catenin pathway 7 . Several genetic mutations observed in ASD are linked with the dysregulation of the WNT/β-catenin pathway through interactions between chromodomain helicase DNA binding protein 8 (CHD8) and CTNNB1 (β-catenin) 8 . In many ASD findings, the WNT/β-catenin pathway is increased 7,[9][10][11] .
Circadian clocks have a main role in some tissues by driving circadian expression of genes involved in physiologic and metabolic functions 12 . One of the key integrator of these complex mechanisms is the canonical WNT/ β-catenin pathway 13,14 . ASD progression is associated with a major metabolic reprogramming, initiated by aberrant WNT/β-catenin pathway, the aerobic glycolysis 11 . In parallel, the dysregulation of circadian rhythms (CRs) upregulates the WNT/β-catenin pathway 15 , which in turn participates to the ASD initiation. This review focuses on the interest of CRs dysregulation in metabolic reprogramming in ASD through the aberrant upregulation of the canonical WNT/β-catenin pathway.

Circadian rhythms
CRs are major biological phenomena found in all universal processes. Their endogenous characteristic is an innate oscillation associated with a period of over 1 day. All the studied organisms show this oscillatory process. Numerous cell functions present temporal variations driven by these oscillatory and circadian ways, including gene expression, metabolic reprogramming, and molecular and cellular pathways. Different integration levels allow the study of the CRs, as endocrinal, physiological, neuronal cell behaviors. Although the coordination and the modulation of CRs are organized by specific pacemaker structures, the primary circadian oscillations are controlled at the cell level. These oscillations are determined by numerous clock genes 16 . The control of the circadian clock is based on an intracellular temporal tracking system that allows anterior organisms to change direction and thus adapt their behavior and the physiology of their life span 17 . It is well known that in many animals species, circadian clock is formed by a specific set of transcription factors which constitutes its molecular architecture. These determinants are both used in positive and negative feedback, which are modulated through a cell-autonomous manner 18 .
Endogenous oscillations generate a freewheeling period, which is close to 24 h, to maintain for the organism constant ambient conditions. These oscillators, at the molecular level, are based on the products of clock regulator genes organized in a transcriptional feedback loop. Circadian oscillations are the product of these posttranscriptional modifications of proteins 19 . A complex loop operates with clock gene transcriptional activators and in turn clock genes with a negative feedback role inhibiting their expression by disrupting the activity of their activators 20 . Several input pathways involve environmental information which interacts with the different compounds of the oscillators. The oscillators are synchronized with the 24 h solar day. The input pathways generate a day-time to transpose it by the oscillators to the output pathways. These output pathways control and regulate the expression of circadian clock genes to generate the rhythmicity. Moreover, the output pathways are predicted to be rhythmic and then controlled by the clock gene transcription factors. These compounds, in turn, regulate downstream the circadian clock genes in a timeof-day-specific manner 21 . This system can synchronize with its environmental time by its internal clock. To respect the environment, the input pathways are vital to maintain this timing for oscillators. A the process named entrainment, the input pathways can reset the activity of the oscillators to stay in a conform 24-h period of the environment 21 . Environmental cues can be detected by input pathways which in turn can modulate many mechanisms to control the activity or level of compounds of oscillators to keep a correct time-of-day expression. This phenomenon is observed in several environmental cues, including nutrition, social interactions and temperature 22,23 . Furthermore, the clock allows a strategy named gating to restrict responses to environmental cues at some day times. Diurnal mammals are insensitive to a light pulse during the day. Nevertheless, during the night, a light pulse can advance or delay the clock to synchronize diurnal mammals with the environment 18 . Environmental signals can interact with molecular oscillators in some cells in complex multicellular organisms. In unicellular organisms, each cell is modulated by oscillators in response to light 24 . However, in multicellular organisms, only a part of the cells has sensory capabilities leading to clock oscillators. The oscillators, and thus the overall rhythmicity of organisms, are concentrated into compounds including a master pacemaker and peripheral oscillators 25 . Face to these sensory inputs, the organism presents some nervous systems which possess environmental cue abilities as a central oscillators or pacemakers rather than to individual cells. In humans, the sensory clock inputs are localized in the brain, where signals from the master pacemaker leads to oscillators in some tissues of the organism.
Nonvisual retinal ganglion cells receive and perceive the light, and transmit this information to the master pacemaker (localized in the hypothalamus) through neural connections. The central pacemaker synchronizes oscillators to the other tissues by using circadian input pathways from the nervous system to peripheral cell systems. Moreover, to maintain the entrainment of these peripheral oscillators by the environment, this central system ensures that cellular oscillations within tissues are properly in phase to provide resonance between individual cellular rhythms 6 . Melatonin operates as a major synchronizer in humans and provides temporal feedback to oscillators within the nervous system to control the circadian phase and the stability of the rhythm 26 . In humans, as in other mammals, melatonin is considered as the main influencer of CRs through its action on receptors in the nervous system 27 .
The sleep-wake pattern is controlled by both CRs and homeostasis. Sleep pressure has been enhanced during the phase of waking and then decreases during the phase of sleeping. The sleep-wake pattern is controlled by the cycle of light-darkness 28 . Through a feedback, sleep-wake pattern can also control the CRs. For many studies, this pattern can be defined as an interface between environmental information (social, mood and cognition) and CRs 29 . Moreover, these two patters are influenced by melatonin 4 . Melatonin level is modulated by the light-dark exposure. Melatonin is associated with sleep initiation. Mutations in Clock, Bmal1, Cry1, Cry2 genes lead to the initiation of some alterations in sleep time and sleep fragmentation 30,31 .

Circadian clock
Some biological mechanisms in humans are controlled by the circadian "clock" (circadian locomotors output cycles kaput) (Fig. 1). The circadian clock is localized in the hypothalamic suprachiasmatic nucleus (SCN). CRs are endogenous and entrainable free-running periods that last 24 h. Several transcription factors can control and modulate CRs. These factors are named circadian locomotor output cycles kaput (Clock), brain and muscle arylhydrocarbon receptor nuclear translocator-like 1 (Bmal1), Period 1 (Per1), Period 2 (Per2), Period 3 (Per3) and Cryptochrome (Cry1 and Cry2) 32,33 . They are modulated by positive and negative self-loop-regulation mediated by CRs 18,34 . Clock and Bmal1 heterodimerize and lead to the transcription of Per1, Per2, Cry1 and Cry2 (ref. 35 ). The Per/Cry heterodimer inhibits its activation by a negative feedback. It translocates back to the nucleus to directly downregulate the Clock/Bmal1 complex and then inhibits its own transcription 35 . The Clock/Bmal1 heterodimer activates the transcription of retinoic acid-related orphan nuclear receptors, Rev-Erbs and retinoid-related orphan receptors (RORs). Through a positive feedback loop, RORs activate the transcription of Bmal1, whereas through a negative feedback loop, Rev-Erbs downregulate their transcription 35 .

Circadian clocks in ASD
Sleep disorders are frequently associated with ASD ( Fig. 2) 36 . Patients suffering from ASD are more correlated with falling asleep anxiety, sleep disorders and CRs disturbance 36 . Moreover, the prevalence of sleep disturbance is associated with cognitive impairments [36][37][38] . In parallel, melatonin levels have been dysregulated in ASD patients 4 . Melatonin diurnal levels are decreased as well as melatonin nocturnal levels, showing a global decrease in melatonin production in ASD children and a dysregulation of day-night rhythm [39][40][41][42] . Deregulation in the production of melatonin in ASD patients has been observed due to HIOMT (hydroxiindole O-methyltransferase) deficiency 43 . Recent studies have shown a potential interest in melatonin therapy in the treatment of sleep onset disorder for ASD patients 44 . Significant allelic correlation has been observed for clock genes, including Per1, and other nucleotids [45][46][47] . Genes involved in ASD pathogenesis are part of pathway networks enhanced in synapse formation, including Neuroxin-Neuroligin genes and in the alteration of the balance excitation-inhibition 48 . Parvalbumin expressing interneurons play a main role in ASD initiation. The knockout of Parvalbumin is associated with core symptoms of ASD patients 49 . CRs dysregulation may downregulate the maturation of Parvalbumin cells and then the timing of critical period of plasticity 50 . The dysregulation of CRs could impact the temporal organization of brain maturation and could have a cascade effect on several brain functions. Negative environmental conditions (sleep deprivation, stress…) could impact the CRs and thus redox homeostasis and transcriptional control of Parvalbumin genes involved in synapse formation and maturation of brain functions.

Melatonin and ASD
Melatonin plays a main role in neurodevelopment 51 . Disturbances in sleep-wake rhythm in ASD could be due to the dysregulation of melatonin 52 . Sleep latency and nocturnal and early awakenings have been reported in ASD 41 . In ASD, the nocturnal secretion of melatonin is low 53 . Intellectual disabilities are associated with melatonin abnormalities 54 . However, in the Down syndrome, the production of melatonin is normal whereas melatonin production is increased in the Fragile X patients 55 . Several therapeutic studies have focused on the interest of melatonin strategy in ASD 56 . Melatonin use in ASD children is associated with improvement of communication 57 , stereotyped behaviors 58 , anxiety 59 and social withdrawal 58 . Nevertheless, melatonin is influenced by age and pubertal stage 60 , and in these studies the autistic behavioral impairment was generally enough described 57 .

Aerobic glycolysis
In mammalian cells, glucose is the main source of energy. All tissues need ATP to function normally. Cells produce ATP by a careful drop in oxidation state from energy-rich molecules like glucose, through the cell respiration process, down to product CO 2 at the end. This process occurs in an aerobic or anaerobic manner, depending on whether oxygen is available. Glycolysis is the first step in glucose metabolism signaling and occurs in the cytosol of all cells. The presence of O 2 is important because of oxidation of glucose under aerobic conditions results in~32 molecules of ATP per molecule of glucose. Under anaerobic conditions, only two molecules of ATP can be produced. Aerobic glycolysis can occur in these two stages. The first occurs in the cytosol and involves the conversion of glucose to pyruvate resulting in NADH production and generating two molecules of ATP. In normal conditions, when oxygen is available, the energy contained in NADH is further released via re-oxidization of the mitochondrial chain and leads to the release of 30 molecules of ATP per molecule of glucose. Pyruvate is reduced to lactate, instead of re-oxidized, under aerobic glycolysis 61 . Thus, glucose is metabolized in order to produce ATP, by a cytosolic glycolysis and oxygendependent mitochondrial physiological mechanism. Glucose entry into the tricarboxylic acid (TCA) cycle is modulated by pyruvate dehydrogenase complex (PDH) 62 . Pyruvate is oxidized to acetyl-coA in mitochondria by the PDH. Acetyl-coA translocates to the TCA cycle for oxidation. Under aerobic glycolysis, pyruvate is converted into lactate in the cytosol by lactate dehydrogenase A (LDH-A). Moreover, aerobic glycolysis is caused by the involvement of hexokinase 2 (HK2) instead of HK1 and pyruvate kinase M2 (PKM2) instead of PKM1 (ref. 63 ). This phenomenon is called aerobic glycolysis or the Warburg effect. Fig. 2 Circadian rhythms and autism spectrum disorder. Relationship between ASD, circadian rhythms and sleep disturbance. Alterations in clock genes and melatonin pathway contribute to the dysregulation of circadian sleep rhythmicity. Circadian rhythms deregulation leads to brain metabolism alterations contributing to ASD. In a negative feedback, ASD symptomatology reinforces circadian rhythms and sleep disturbances creating a self-reinforcing circle.
Pyruvate production is stimulated 65,67 , but with an increased ratio of lactate-to-pyruvate 65,68 showing a high glucose metabolism and LDH-A activity in ASD patients. Moreover, LDH-A expression has been increased in ASD patients 71 . A recent study has presented a decrease level of pH correlated with high levels of lactate in ASD patients 72 . These observations could suggest an increase of aerobic glycolysis in ASD since the dysregulation of this balance has been proposed as a candidate cause of ASD 73 .

CRs and aerobic glycolysis
Few studies have focused on the relationship between CRs and aerobic glycolysis (Fig. 2). Nevertheless, this relation could be mainly interesting in the development of tumors 74 . In the same way, melatonin expression and modulation by CRs in cancers is associated with the disruption of the aerobic glycolysis [75][76][77] . Thermodynamic and energy reprogramming highlight this relation in fibrosis 78 , neurodegenerative diseases 79,80 and cancers 81 . The importance of 24-h fluctuations in the aerobic glycolysis and the availability of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) in cancer has been shown through the consideration of the redox influence on NADPH 82 .

The canonical WNT/β-catenin pathway
The Wingless/Int (WNT) pathway is a family of secreted lipid-modified glycoproteins (Fig. 3) 83 . Several nervous molecular mechanisms are modulated by the WNT/ β-catenin pathway, including development of synapses in the central nervous system 84,85 , synaptogenesis 86,87 and control of synaptic formation 84,88 . Numerous pathophysiologic signalings are mediated by the dysregulation of this pathway, including cancers 89,90 , fibrosis 91 , neurodegenerative diseases 80 and angiogenesis 13,92 . Stimulation of β-catenin signaling needs the presence of the complex LRP5 /LRP6 93 . LRP5 has a main role while LRP6 presents a minor role in the retinal vascularization 94,95 . Disheveled (Dsh) forms a complex with Axin, and this prevents the phosphorylation of β-catenin by glycogen synthase kinase-3β (GSK-3β). Then, β-catenin accumulation in the cytosol is observed and translocates to the nucleus to bind T-cell factor/lymphoid enhancer factor (TCF/LEF) cotranscription factors. This nuclear binding allows the transcription of WNT-responsive genes, such as cyclin D1, c-Myc, PDK1, MCT-1 (refs. 96,97 ). WNT ligands' absence is associated with cytosolic β-catenin phosphorylation by GSK-3β.
Phosphatase and tensin homolog protein (PTEN) inhibition is associated with the increase of the WNT pathway and high risk of ASD development [124][125][126] . In Purkinje cells, PTEN inhibition impairs social relation, behavior and deficits in motor learning 127,128 . PTEN and β-catenin control each other to normal brain growth and development 129 .

CRs and WNT/β-catenin pathway
RORs are upstream effectors of the WNT/β-catenin pathway 130 . By this interaction, circadian genes can modulate the cell cycle progression 131 . A Bmal1 knockdown can downregulate the WNT/β-catenin pathway 132 . In wild-type mice, the levels of WNT-related genes are higher than those observed in Bmal1 knockdown mice 133,134 . The proliferation and progression of cell cycle are controlled by Bmal1 by activating the WNT/β-catenin pathway 135 . Bmal1 involves the β-catenin transcription, diminishes the β-catenin degradation and then inhibits GSK-3β activity 136 . In the intestinal mucosa of ApcMin/+ mice, the degradation of Per2 leads to β-catenin increase by circadian disruption 137 .
In normal conditions, the core circadian genes operate in accurate feedback loops and keep the molecular clockworks in the SCN. They allow the control of peripheral clocks 18,34 . Per1 and Per2 maintain cell CRs and modulate cell-related gene activity, such as c-Myc, so as to sustain the physiologic cell cycle 138,139 .

Aerobic glycolysis and WNT/β-catenin pathway
Some reports have highlighted that the WNT/β-catenin pathway is closely associated and a main effector of the aerobic glycolysis (Fig. 4) 11,78,[140][141][142] . The PI3K/Akt pathway stimulates the glucose metabolism to enhance protein and lipid synthesis 143 . Moreover, PI3K/Akt pathway increases the glucose metabolism to protect cells against reactive oxygen species (ROS) stress induced by activated HIF-1α and decreasing the glucose entry into the TCA cycle 144 . HIF-1α stimulates pyruvate dehydrogenase kinase (PDK) to phosphorylate PDH and inactivates it, leading to cytosolic pyruvate being shunted into lactate by LDH-A 145 . HIF-1α is transcriptionally activated by PI3K/Akt/mTOR pathway through 4E-BP1 and STAT3 (refs. [146][147][148][149][150][151]. Numerous studies have observed that the WNT/ β-catenin pathway can downregulate the pyruvate oxidation in the TCA cycle 140,152 . WNT/β-catenin pathway, by activating both the PI3K/Akt/mTOR pathway and HIF-1α, can lead to aerobic glycolysis 140,152,153 . PI3K/Akt pathway can also control the β-catenin accumulation and then the expression of the downstream genes 154 . c-Myc directly stimulates the HIF-1α 155 , PDK and lactate transporter (MCT-1) expressions 152 . The stimulation of HIF-1α leads to the overexpression of glucose Fig. 3 The canonical WNT/β-catenin pathway. Inactivated WNT: Under physiologic circumstances, the cytoplasmic β-catenin is linked to its destruction complex, consisting of APC, AXIN and GSK-3β. β-catenin is phosphorylated by GSK-3β. Thus, phosphorylated β-catenin is destroyed in the proteasome. Then, the cytoplasmic level of β-catenin is kept low in the non-presence of WNT ligands. If β-catenin is not accumulated in the nucleus, the TCF/LEF complex does not stimulate the target genes. DKK1 inhibits the WNT/β-catenin pathway through binding to WNT ligands or LRP5/6. Activated WNT: When WNT ligands activate both FZD and LRP5/6, DSH is stimulated and phosphorylated by FZD. Phosphorylated DSH in turn activates AXIN, which comes off the β-catenin destruction complex. Thus, β-catenin escapes from phosphorylation and then accumulates in the cytoplasm. The accumulated cytosolic β-catenin moves into the nucleus, where it interacts with TCF/LEF and stimulates the transcription of target genes. transporters (Glut), hexokinase (HK), pyruvate kinase (PK), PDK1 and LDH-A [156][157][158][159] .

Conclusion
Changes in energy metabolism are modulated by abnormal CRs in ASD patients. In ASD, the canonical WNT/β-catenin pathway is increased. Energy behaviors of metabolic enzymes in ASD are modified by this upregulation of the WNT/β-catenin pathway leading to the enhancement of aerobic glycolysis and thus the production of lactate. This explains the glucose hypermetabolism observed in ASD. WNT pathway is driven by the CRs and operates under a circadian regime evolving to changes in energy metabolism. CRs directly contribute to the regulation of the molecular pathways WNT/ β-catenin pathway involved in the reprogramming of cellular energy metabolism enabling ASD.  Interactions between WNT pathway and energy metabolism in ASD. In ASD, the WNT pathway is activated. In the presence of WNT ligands, cytosolic β-catenin is accumulated in cytosol and GSK-3β is inhibited. APC and Axin combine with GSK-3β and DSH to form a complex with LRP 5/6 and FZD. β-catenin translocates to the nucleus and binds to TCF/LEF co-transcription factor. WNT target genes, such as cMyc, are activated. β-catenin accumulation increases the level of PI3K/Akt pathway and results in the activation of HIF-1α. Activated HIF-1α stimulates Glut, HK, PKM2, LDH-A and PDK1. Activation of HIF-1α involves PKM2 translocation to the nucleus. PKM2 activates PEP cascade and the formation of pyruvate. PKM2 binds to β-catenin and induces cMyc-mediated expression of glycolytic enzymes (Glut, LDH-A, PDK1). Activation of Glut and HK involves glucose hyper-metabolism with increase in glucose transport and phosphorylation rates. PDK1 inhibits PDH to downregulate the pyruvate entrance into mitochondria. Lactate production is activated by LDH-A. This is aerobic glycolysis.