CHOLESTEROL METABOLISM IN AUTISM SPECTRUM DISORDERS

Abstract

Autism spectrum disorders (ASDs) represent a range of neurodevelopmental pathologies, characterized by impaired social interactions, delay of cognitive functions, compromised communication, repetitive and restricted behaviors, and comorbid features (Faras et al., 2010). Usually these disabilities affect children during childhood, and present different phenotypes, considering the multi-factorial nature of ASDs. In fact, despite genetic alterations play a role in the development of these disorders, the role of environmental factors, during prenatal life, is becoming an important area of research to find other aspects not yet analyzed, but could be very important to clarify the autism etiology. Interestingly, ASDs have been associated to a loss of cholesterol homeostasis (Tierney, 2008; Wang, 2014). Cholesterol is an important lipid, synthesized by 3β-hydroxy 3β-methylglutharyl Coenzyme A reductase (HMGCR) through the Mevalonate (MVA) pathway that produces, beside cholesterol, other important compounds such as prenyls, dolichol, and ubiquinone. Remarkably, cholesterol homeostasis displays a sex- and age-dependent regulation both in liver and in Central Nervous System (CNS) (De Marinis et al., 2008; Segatto et al., 2013). Moreover, although cholesterol homeostasis in the CNS is regulated by the same protein network operating in peripheral tissues, it presents some peculiarities: i) the cholesterol present into the CNS is completely separated from the rest of the body, thus it is synthesized in situ; ii) prenatally in mammals, during brain development, neurons are able to synthesize cholesterol, while postnatal neurons reduce or abandon cholesterol synthesis up-taking it from astrocytes (Martin et al., 2014; Segatto et al., 2014). As far as we know, it is not clear if the reduction of cholesterol synthesis in neurons is a cause or a consequence of neuronal differentiation. Moreover, despite a relation between the neurodevelopmental disorders such as ASDs and cholesterol has been hypothesized, no information are present about MVA pathway and cholesterol homeostasis in a preclinical model of ASDs. For these reasons, the mains goals of my PhD project have been: 1) To study the role of MVA pathway and the modulation of the protein network of cholesterol homeostasis during neuronal differentiation; 2) To study, in an ASDs experimental model, the protein network of cholesterol homeostasis in different brain areas at different age of development; 3) To identify whether sex-related differences are present in the same experimental model of autism. To start, using differentiated N1E-115 cell line as in vitro experimental model, we analyzed the protein network regulating MVA pathway. The results demonstrated that HMGCR levels decreased during neuronal differentiation, suggesting that a physiological downregulation of MVA pathway activation occurs during. HMGCR inhibition by Simvastatin (Sim) supports this notion, as it determined the increase of neurite elongation. Interestingly, this effect was related to a reduced RhoA prenylation/activation. Overall, these data underlined a key role of MVA pathway in neuronal differentiation, and indicate that the decrease of MVA pathway activity is a cause rather than an effect of this physiological process. The findings obtained by N1E-115 let us suppose that alterations in MVA pathway could be involved in the onset of neurodevelopmental disorders, such as autism spectrum disorders (ASDs). In order to delve deeper into this hypothesis, we analyzed the protein network controlling MVA pathway in infant, adolescent and adult male rats prenatally exposed to valproic acid (VPA), a well-established experimental model of autism (Nicolini and Fahnestock, 2017). We found that prenatal VPA exposure did not alter cholesterol metabolism in both plasma and liver of VPA-animals. On the other hand, disruptions in cholesterol/isoprenoid homeostasis hippocampus, prefrontal cortex, cerebellum, amygdala, dorsal striatum and nucleus accumbens were present in the CNS. In particular, VPA exposure determined a peculiar and distinctive impairment for each brain region. For instance during adolescence, the most affected period by VPA exposure, evident alterations in geranylgeranylated and farnesylated protein levels were observed in the cerebellum, nucleus accumbens and striatum; whereas decreased cholesterol levels were only highlighted in the hippocampus, probably depending on the reduced number of olygodendrocytes found in this brain region. The incidence of autism, as well as the regulation of cholesterol/isoprenoid metabolism, display typical differences between both sexes. Thus, in the attempt to find additional correlations between autism and perturbations in cholesterol/isoprenoid homeostasis, we verified whether sex-related differences were also present in adolescent and adult male and female rats prenatally exposed to VPA. Results demonstrated that VPA specifically affected cholesterol/isoprenoid metabolism in the brain, in an age and sex-dependent manner. Taken together, the results obtained during my PhD studies underlie the pivotal role of MVA pathway in neuronal development, suggesting that any interference in this process could induce behavioral alterations like those ones occurring in ASDs. Moreover, these results suggest for the first time, a possible correlation among cholesterol homeostasis, ASDs and sex.