Anorexia Nervosa (AN) is a devastating psychiatric disorder affecting pubescent females ninefold more than males, in a period of life extremely vulnerable to external stimuli: the adolescence. AN begins with a self-induced restrictive diet to lose weight, below 85% of expected body mass index, that in combination with intense physical activity and aberrant body image concern, it progresses to an out-of-control spiral. In this condition, the positive experience of control on food intake and intense exercise is indeed extremely rewarding for the patient, reinforcing the dieting behavior. AN is associated with the highest mortality rate among the wide family of psychiatric disorders, and despite the knowledge about the clinical symptomatology, AN etiopathogenesis remains unclear, treatment is challenging and often hampered by high relapse. At the neurobiological level, patients suffering from AN display altered neural activity, morphological and functional connectivity in the fronto-striatal and thalamo-cortical circuits. In particular, hypoglutamatergic transmission and aberrant excitability of cortical and striatal regions observed in AN patients might underpin cognitive deficits that fuel the vicious cycle of dieting behavior. It has been suggested that the driving force of such abnormal behavior might be represented by an altered balance between reward and inhibition mechanisms in the brain, the first mainly mediated by the Nucleus Accumbens (NAc), and the latter being normally triggered by the medial prefrontal cortex (mPFC) that is immature and still growing during adolescence. Such dysfunctional mechanisms combined with alterations in the levels of appetite modulators, such as ghrelin and leptin, and dysregulations in peripheral organs and metabolic active tissues functionality may lead to a distorted response to salient stimuli, such as food, fueling the maintenance of the anorexic phenotype. AN is not only a brain disorder but it is rather a systemic disease that affects the whole body, particularly skeletal muscles and circulating hormones, suggesting the presence of a crosstalk between peripheral signals and brain functions. Therefore, the main goal of this project is to investigate from a preclinical point of view the neurobiological mechanisms involved in the pathophysiology of AN. We pointed our attention to the possibility that the combination of food restriction with intense exercise, the hallmarks of AN, activates dysfunctional periphery-to-brain crosstalk that may drive for weight loss seeking inducing aberrant dieting behaviors, affecting reward processes and cognition, via altered neurometabolic pathways in specific areas of the brain, and in turn, promoting the long-lasting maintenance of AN disorder. Therefore, with the aim to investigate the central and peripheral alterations in biological mechanisms that may support the anorexic condition and induce cognitive impairments, we employed the well-known animal model of AN, the activity-based anorexia (ABA) rat model, which recapitulates human AN by combining caloric restriction and physical exercise to induce self-induced body-weight loss. We have demonstrated that, under a paradigm of food restriction and free access to a mechanical activity wheel, adolescent female rats showed reduced food intake, reduced body weight and increased running activity on the wheel, thus developing the anorexic phenotype. To dissect the neurobiological underpinnings of this condition and the cognitive-related dysfunctions during a critical developmental period, we performed structural, morphological and molecular studies on critical brain areas of the reward system, crucially involved in processing feeding information and in mediating high-level cognitive functions such as the NAc and the mPFC. In both AN patients and animal models of AN, evidence exists of glutamate homeostasis dysfunctions, which has been proposed as a signal of altered processing of food reward. Thus, we firstly focused our attention on critical determinants of the glutamatergic synapse evaluating AMPA, NMDA receptors, their scaffolding proteins and their localization at the post-synaptic density, dendritic spines density and morphology, and lastly cognitive-related outcomes. Furthermore, based on the hypothesis that the energy imbalance, via altered regulation of molecules released by peripheral organs, can lead to the alteration of neurocircuits responsible for motivational and cognitive processes, we performed a neurometabolic analysis of the PGC1/FNDC5/Irisin/BDNF pathway, linking molecular alterations in skeletal muscle with alterations in the hippocampus (Hip), respectively involved in physical activity and in memory and learning processes. All the molecular analysis has been performed at two time points: early after the induction of the anorexic phenotype at post-natal day (PND) 42, which mimics the acute phase of the pathology, and after a period of body weight recovery at PND49, in order to investigate both the alterations induced by the AN acute phase and the potential long-lasting alterations that persist even after the body weight recovery period. Overall, our analysis revealed that the combination of a restricted regimen of food intake and free wheel access induced a general dysregulation of the glutamatergic homeostasis in both NAc and mPFC regions. We observed AMPA and NMDA receptor subunit reorganization and retention in different subcellular fractions, paralleled with structural changes in dendritic spines density and morphology and altered temporal memory performance. Of note, all these alterations persist even after the body weight recovery. Interestingly, the analysis of the neurometabolic axis on the PGC1/FNDC5/Irisin/BDNF pathway revealed an initial hyperactivation of the axis induced by the induction of the acute phase of AN, as shown by increased PGC1 and FNDC5 protein levels in muscle tissues and increased irisin plasma levels observed at PND42. In the Hip, BDNF transcription was increased, while protein translation was reduced specifically in ABA rats. After body weight recovery, despite PGC1 and FNDC5 protein levels were normalized, BDNF expression in the Hip was persistently reduced, suggesting its putative involvement, not only in altering the hippocampal neuroplasticity, but also in the induction of associative reward learning alterations that may underpins the observed long-lasting memory deficits. In conclusion, the obtained results point toward the reorganization of the glutamatergic synapse structure and composition in crucial brain areas of the reward system, as critical factor in driving the motivational mechanisms that fuel the maladaptive behaviors underlying AN. Moreover, our data provides novel insights in the involvement of a periphery-to-brain neurometabolic crosstalk even when body weight is restored. These molecular determinants of maladaptive plasticity could represent a signal of altered processing of food reward, and a vulnerability trait for relapse. Moreover, the herein shown AN-induced dysregulation of the glutamatergic system and hippocampal BDNF system coupled with an altered metabolic profile might be the trigger that could lead, in turn, to cognitive dysfunction, which is consistently observed in AN patients.

MALADAPTIVE PLASTICITY IN ANOREXIA NERVOSA: EVIDENCE OF A PERIPHERY-TO-BRAIN CROSSTALK IN A PRECLINICAL MODEL / F. Mottarlini ; tutor: F. Fumagalli; coordinator: G. D. Norata. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Dec 09. ((34. ciclo, Anno Accademico 2021.

MALADAPTIVE PLASTICITY IN ANOREXIA NERVOSA: EVIDENCE OF A PERIPHERY-TO-BRAIN CROSSTALK IN A PRECLINICAL MODEL

F. Mottarlini
2021

Abstract

Anorexia Nervosa (AN) is a devastating psychiatric disorder affecting pubescent females ninefold more than males, in a period of life extremely vulnerable to external stimuli: the adolescence. AN begins with a self-induced restrictive diet to lose weight, below 85% of expected body mass index, that in combination with intense physical activity and aberrant body image concern, it progresses to an out-of-control spiral. In this condition, the positive experience of control on food intake and intense exercise is indeed extremely rewarding for the patient, reinforcing the dieting behavior. AN is associated with the highest mortality rate among the wide family of psychiatric disorders, and despite the knowledge about the clinical symptomatology, AN etiopathogenesis remains unclear, treatment is challenging and often hampered by high relapse. At the neurobiological level, patients suffering from AN display altered neural activity, morphological and functional connectivity in the fronto-striatal and thalamo-cortical circuits. In particular, hypoglutamatergic transmission and aberrant excitability of cortical and striatal regions observed in AN patients might underpin cognitive deficits that fuel the vicious cycle of dieting behavior. It has been suggested that the driving force of such abnormal behavior might be represented by an altered balance between reward and inhibition mechanisms in the brain, the first mainly mediated by the Nucleus Accumbens (NAc), and the latter being normally triggered by the medial prefrontal cortex (mPFC) that is immature and still growing during adolescence. Such dysfunctional mechanisms combined with alterations in the levels of appetite modulators, such as ghrelin and leptin, and dysregulations in peripheral organs and metabolic active tissues functionality may lead to a distorted response to salient stimuli, such as food, fueling the maintenance of the anorexic phenotype. AN is not only a brain disorder but it is rather a systemic disease that affects the whole body, particularly skeletal muscles and circulating hormones, suggesting the presence of a crosstalk between peripheral signals and brain functions. Therefore, the main goal of this project is to investigate from a preclinical point of view the neurobiological mechanisms involved in the pathophysiology of AN. We pointed our attention to the possibility that the combination of food restriction with intense exercise, the hallmarks of AN, activates dysfunctional periphery-to-brain crosstalk that may drive for weight loss seeking inducing aberrant dieting behaviors, affecting reward processes and cognition, via altered neurometabolic pathways in specific areas of the brain, and in turn, promoting the long-lasting maintenance of AN disorder. Therefore, with the aim to investigate the central and peripheral alterations in biological mechanisms that may support the anorexic condition and induce cognitive impairments, we employed the well-known animal model of AN, the activity-based anorexia (ABA) rat model, which recapitulates human AN by combining caloric restriction and physical exercise to induce self-induced body-weight loss. We have demonstrated that, under a paradigm of food restriction and free access to a mechanical activity wheel, adolescent female rats showed reduced food intake, reduced body weight and increased running activity on the wheel, thus developing the anorexic phenotype. To dissect the neurobiological underpinnings of this condition and the cognitive-related dysfunctions during a critical developmental period, we performed structural, morphological and molecular studies on critical brain areas of the reward system, crucially involved in processing feeding information and in mediating high-level cognitive functions such as the NAc and the mPFC. In both AN patients and animal models of AN, evidence exists of glutamate homeostasis dysfunctions, which has been proposed as a signal of altered processing of food reward. Thus, we firstly focused our attention on critical determinants of the glutamatergic synapse evaluating AMPA, NMDA receptors, their scaffolding proteins and their localization at the post-synaptic density, dendritic spines density and morphology, and lastly cognitive-related outcomes. Furthermore, based on the hypothesis that the energy imbalance, via altered regulation of molecules released by peripheral organs, can lead to the alteration of neurocircuits responsible for motivational and cognitive processes, we performed a neurometabolic analysis of the PGC1/FNDC5/Irisin/BDNF pathway, linking molecular alterations in skeletal muscle with alterations in the hippocampus (Hip), respectively involved in physical activity and in memory and learning processes. All the molecular analysis has been performed at two time points: early after the induction of the anorexic phenotype at post-natal day (PND) 42, which mimics the acute phase of the pathology, and after a period of body weight recovery at PND49, in order to investigate both the alterations induced by the AN acute phase and the potential long-lasting alterations that persist even after the body weight recovery period. Overall, our analysis revealed that the combination of a restricted regimen of food intake and free wheel access induced a general dysregulation of the glutamatergic homeostasis in both NAc and mPFC regions. We observed AMPA and NMDA receptor subunit reorganization and retention in different subcellular fractions, paralleled with structural changes in dendritic spines density and morphology and altered temporal memory performance. Of note, all these alterations persist even after the body weight recovery. Interestingly, the analysis of the neurometabolic axis on the PGC1/FNDC5/Irisin/BDNF pathway revealed an initial hyperactivation of the axis induced by the induction of the acute phase of AN, as shown by increased PGC1 and FNDC5 protein levels in muscle tissues and increased irisin plasma levels observed at PND42. In the Hip, BDNF transcription was increased, while protein translation was reduced specifically in ABA rats. After body weight recovery, despite PGC1 and FNDC5 protein levels were normalized, BDNF expression in the Hip was persistently reduced, suggesting its putative involvement, not only in altering the hippocampal neuroplasticity, but also in the induction of associative reward learning alterations that may underpins the observed long-lasting memory deficits. In conclusion, the obtained results point toward the reorganization of the glutamatergic synapse structure and composition in crucial brain areas of the reward system, as critical factor in driving the motivational mechanisms that fuel the maladaptive behaviors underlying AN. Moreover, our data provides novel insights in the involvement of a periphery-to-brain neurometabolic crosstalk even when body weight is restored. These molecular determinants of maladaptive plasticity could represent a signal of altered processing of food reward, and a vulnerability trait for relapse. Moreover, the herein shown AN-induced dysregulation of the glutamatergic system and hippocampal BDNF system coupled with an altered metabolic profile might be the trigger that could lead, in turn, to cognitive dysfunction, which is consistently observed in AN patients.
FUMAGALLI, FABIO
NORATA, GIUSEPPE DANILO
Settore BIO/14 - Farmacologia
MALADAPTIVE PLASTICITY IN ANOREXIA NERVOSA: EVIDENCE OF A PERIPHERY-TO-BRAIN CROSSTALK IN A PRECLINICAL MODEL / F. Mottarlini ; tutor: F. Fumagalli; coordinator: G. D. Norata. - : . Dipartimento di Scienze Farmacologiche e Biomolecolari, 2021 Dec 09. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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