The role of N6-methyladenosine RNA methylation in the crosstalk of circadian clock and neuroinflammation in rodent suprachiasmatic nuclei.

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

Streptozotocin-Induced Astrocyte Mitochondrial Dysfunction Is Ameliorated by FTO Inhibitor MO-I-500.

The pathogenesis of Alzheimer's disease (AD), the most prevalent form of dementia, remains unclear. Over the past few years, evidence has accumulated indicating that perturbed cerebral bioenergetics and neuroinflammation may compromise cognitive functions and precedes the onset of AD and that impaired function of glial cells can likely contribute to the development of the disease. Recently, N6-methyladenosine (m6A) modification of RNA has been implicated in the regulation of different processes in the brain and to play a potential role in neurodegeneration. In the present study, we investigated the potential role of the m6A machinery enzymes in a streptozotocin (STZ) model of AD in human astrocytoma CCF-STTG1 cells. We observed that STZ-treated astrocytes expressed significantly higher levels of m6A demethylase fat mass and obesity-associated protein (FTO) and m6A reader YTHDF1 (YTH domain-containing family protein 1). Our experiments revealed that MO-I-500, a novel pharmacological inhibitor of FTO, can strongly reduce the adverse effects of STZ. Inhibition of FTO enhanced the survival of cells exposed to STZ and suppressed oxidative stress, apoptosis, elevated expression of glial fibrillary acidic protein, mitochondrial dysfunction, and bioenergetic disturbances induced by this compound. Overall, the results of this study indicate that perturbed m6A signaling may be contributing to AD pathogenesis, likely by compromising astrocyte bioenergetics.

Prenatal exposure to lipopolysaccharide induces changes in the circadian clock in the SCN and AA-NAT activity in the pineal gland.

The mammalian circadian pacemaker in the suprachiasmatic nucleus (SCN) regulates behavioral and physiological processes in a 24-h cycle. During its development, the SCN can be sensitive to external stimuli which may change the circadian phenotypes in adulthood. Here, we investigated the effects of prenatal exposure to endotoxin lipopolysaccharide (LPS) on the developing rhythms in expression of Per1, Per2, Nr1d1 and Rasd1 along the rostrocaudal axis of the SCN, and on the rhythm of the rate-limiting enzyme in melatonin synthesis, pineal alkylamine N-acetyltransferase (AA-NAT). The prenatal LPS treatment induced anxiety-like behavior in adulthood as shown before and affected the rhythmicity of clock genes in the SCN. The major effect was observed for Nr1d1 expression; the least affected gene was Per2. The Nr1d1 in the LPS-treated group was arrhythmic at postnatal day 3, but showed significantly higher amplitude at postnatal day 20 at all SCN parts, similarly to the AA-NAT activity in pineal glands, thus suggesting adaptive flexibility of the developing SCN to immune challenges in early development.

Protein profiling of SH-SY5Y neuroblastoma cells: The effect of rhein.

4,5-Dihydroxyanthraquinone-2-carboxylic acid (Rhein) has been shown to have various physiological and pharmacological properties including anticancer activity and modulatory effects on bioenergetics. In this study, we explored the impact of rhein on protein profiling of undifferentiated (UC) and differentiated (DC) SH-SY5Y cells. Besides that, the cellular morphology and expression of differentiation markers were investigated to determine the effect of rhein on retinoic acidinduced neuronal cell differentiation. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry we evaluated the changes in the proteome of both UC and DC SH-SY5Y cells after 24 h treatment with rhein. Validation of selected differentially expressed proteins and the assessment of neuronal differentiation markers were performed by western blotting. Proteomic analysis revealed significant changes in the abundance of 15 proteins linked to specific cellular processes such as cytoskeleton structure and regulation, mitochondrial function, energy metabolism, protein synthesis and neuronal plasticity. We also observed that the addition of rhein to the cultured cells during differentiation resulted in a significantly reduced neurite outgrowth and decreased expression of neuronal markers. These results indicate that rhein may strongly interfere with the differentiation process of SH-SY5Y neuroblastoma cells and is capable of inducing marked proteomic changes in these cells.

The role of rat posterior parietal cortex in coordinating spatial representations during place avoidance in dissociated reference frames on a continuously rotating arena (Carousel).

On the Carousel maze, rats are trained to avoid a sector of a circular rotating arena, punishable by a mild electric foot-shock. In the room frame (RF) variant, the punishable sector remains stable relative to the room, while in the arena frame (AF) version, the sector rotates with the arena. The rats therefore need to disregard local olfactory, tactile and self-motion cues in RF condition and distal extra-maze landmarks in the AF task. In both primates and rodents, the coordination of various spatial reference frames is thought to depend on the posterior parietal cortex (PPC). We have previously shown that PPC-lesioned rats can solve both variants of the Carousel avoidance task. Here we aimed to determine the effects of bilateral thermocoagulation lesion of the PPC in Long-Evans rats on the ability to transition between multiple spatial strategies. The rats were first trained in five sessions in one condition and then another five sessions in the other. The following training schemes were used: RF to AF, RF to RF reversal (sector on the opposite side), and AF to RF. We found a PPC lesion-associated impairment in the transition from the AF to RF task, but not vice versa. Furthermore, PPC lesion impaired performance in RF reversal. In accordance to the literature, we also found an impairment in navigation guided by intra-maze visuospatial cues, but not by extra-maze cues in the water maze. Therefore, the PPC lesion-induced impairment is neither specific to distant cues nor to allocentric processing. Our results thus indicate a role of the PPC in the flexibility in spatial behaviors guided by visual orientation cues.

Functional inactivation of the rat hippocampus disrupts avoidance of a moving object.

The hippocampus is well known for its critical involvement in spatial memory and information processing. In this study, we examined the effect of bilateral hippocampal inactivation with tetrodotoxin (TTX) in an "enemy avoidance" task. In this paradigm, a rat foraging on a circular platform (82 cm diameter) is trained to avoid a moving robot in 20-min sessions. Whenever the rat is located within 25 cm of the robot's center, it receives a mild electrical foot shock, which may be repeated until the subject makes an escape response to a safe distance. Seventeen young male Long-Evans rats were implanted with cannulae aimed at the dorsal hippocampus 14 d before the start of the training. After 6 d of training, each rat received a bilateral intrahippocampal infusion of TTX (5 ng in 1 µL) 40 min before the training session on day 7. The inactivation severely impaired avoidance of a moving robot (n = 8). No deficit was observed in a different group of rats (n = 9) that avoided a stable robot that was only displaced once in the middle of the session, showing that the impairment was not due to a deficit in distance estimation, object-reinforcement association, or shock sensitivity. This finding suggests a specific role of the hippocampus in dynamic cognitive processes required for flexible navigation strategies such as continuous updating of information about the position of a moving stimulus.

Inertial stimuli generated by arena rotation are important for acquisition of the active place avoidance task.

The active place avoidance task is used for testing cognitive abilities in rats. A rat, placed on a rotating circular arena, should avoid an unmarked sector defined with respect to stable extra-arena cues. We hypothesized that the inertial stimuli generated by the arena rotation may contribute to the performance in the task. These stimuli provide permanent information to the rat concerning changes in its position with respect to the extra-arena cues, it means to the reference frame in which the to-be-avoided sector is defined. To test the hypothesis, we trained one group of rats on a stable arena while extra-arena cues rotated around the arena. This eliminated the inertial stimuli generated by the arena rotation while preserving other aspects of the task. Six out of seven rats from this group did not learn this modified task. The remaining rat learned it equally well as rats from a control group learned the standard active place avoidance task. After six days of training, we changed the tasks between the groups. The control rats solved the modified task as well as the standard task. We conclude that the inertial stimuli generated by the arena rotation are important for acquisition of the active place avoidance task but not for performance once the task has been mastered. We suggest that rats must perceive the distal extra-arena cues as stable in order to associate the position of the to-be-avoided sector with these cues.

Enemy avoidance task: a novel behavioral paradigm for assessing spatial avoidance of a moving subject.

Navigation with respect to moving goals represents a useful ability in the everyday life of animals. We have developed a novel behavioral paradigm, "enemy avoidance task", in which a laboratory rat (subject) was trained to avoid another rat (enemy), while searching for small pasta pellets dispensed onto an experimental arena. Whenever the distance between the two animals was smaller than 25 cm, the subject was given a mild electric footshock. The results have shown that rats are capable of avoiding another rat while exploring an environment. Therefore, the enemy avoidance task can be used in electrophysiological, lesion or neuropharmacological studies exploring neuronal substrate coding for egocentric and allocentric positions of an observed animal.

Lesion of posterior parietal cortex in rats does not disrupt place avoidance based on either distal or proximal orienting cues.

A current topic in neurobiology is the study of the role of various brain structures in processing of spatial information. The present study was aimed at elucidating the role of the rat posterior parietal cortex in performing a place avoidance task. Two variants of the task were used: an arena frame task, in which animals were trained to avoid a sector defined by local cues bound to the surface of a rotating arena, and the room frame task, in which the shock sector was defined with respect to distal room landmarks. The results showed that both control and lesioned rats were able to efficiently solve both tasks, while locomotion was not altered. These results suggest that the posterior parietal cortex is not crucial for the processing of either proximal or distal cues in place avoidance.

Morris water maze learning in Long-Evans rats is differentially affected by blockade of D1-like and D2-like dopamine receptors.

Dopaminergic neurotransmission is involved in several brain functions including spatial cognition. In the present study we examine the effects of systemic administration of D1-like receptor antagonist SCH23390 and D2-like receptor antagonist sulpiride on the acquisition of the Morris water maze task. We used visible versus hidden platform versions of the MWM in order to distinguish between the effects of the drugs on the procedural versus cognitive aspects of the task. SCH23390 was found to prolong escape latencies to the visible platform at a higher dose (0.05mg/kg), whilst the lower dose (0.02mg/kg) left both procedural and cognitive functions almost unchanged. SCH23390 was also found to reduce swimming speed. Sulpiride did not affect the visible platform learning at any of three doses studied (30, 60 and 100mg/kg); the highest dose of sulpiride (100mg/kg) impaired place navigation to the hidden platform, without affecting the swim speed. The results of the present study show a difference in the involvement of D1-like and D2-like receptors in the MWM acquisition.