Dottorato in FARMACOLOGIA E TOSSICOLOGIA

Titolo della tesi: Reduced availability of sialylated sugars of the human milk during lactation impairs cognitive function in adult mice

Human milk is the ideal food for the newborn. Its composition results from an evolutionary biological process and represents an essential source of nutrients for the baby. Breastfeeding is associated with multiple benefits for both the infant and the mother herself. These findings result from studies comparing phenotypes of breastfed (BF) and formula-fed (FF) infants. Additionally, maternal milk’s components have been shown to persistently adjust the development of cognitive functions. In particular, experimental evidence indicates that breastfed infants show significantly higher intelligence scores compared to FF children. Among the various components of human milk, sialic acid (Sia) has been proposed to exert a pivotal role in mediating the beneficial effects of breastfeeding on the offspring. Sia is a structural molecule of gangliosides and polySia-NCAM, both highly represented in the central nervous system. and involved in mechanisms such as neurogenesis, synaptogenesis as well as the formation of neural circuits. The role exerted by Sia on brain structure in early postnatal stages has been proposed to contribute to the neurological and intellectual advantages conferred by BF over FF. Specifically, human milk represents an essential source of Sia (in the form of sialylated oligosaccharides) whereby the enzyme responsible for the endogenous synthesis of Sia has very low activity in the newborn. Human milk oligosaccharides (HMOs) represent 8% of the sugar’s milk and have a wide range of beneficial effects on the physiology of the infant. Importantly, while HMOs are abundant in human milk, they are scarce in infant formula; finally, preclinical studies indicate that their supplementation may beget substantial cognitive benefits. During my Ph.D., I have investigated the role exerted by sialyl(alpha2,3)lactose (3’SL) and sialyl(alpha2,6)lactose (6’SL) on the development of cognitive capabilities. The selection of 3’SL and 6’SL rests upon the functional role of these sialylated oligosaccharides during development (brain structure patterning, gut microbiota, immunity) and the evidence that, while 3’SL and 6’SL are abundant in maternal milk, they are not provided in infant formula. Based on these considerations, I have tested the hypothesis that a selective absence of 6’SL alone (Chapter 1), 3’SL alone (Chapter 2), and the combination of 6’SL and 3’SL together (Chapter 3) may persistently impair cognitive capabilities (i.e. memory and attention). Then, I evaluated if an exogenous administration of these sugars may ameliorate the affected phenotype (Chapter 3). To do that, I modulate their concentrations leveraging a mouse model characterized by the absence of genes that synthesize 3’SL and 6’SL (B6.129-St3gal4tm1.1Jxm and St6gal1tm2Jxm-J, knock-out mice, hereafter KO), producing milk lacking 3’SL and/or 6’SL. Then, to expose wild-type (WT) pups to 3’SL and/or 6’SL-poor milk, I adopted a cross-fostering protocol in which WT mice were reared to KO dams. In chapter 1, WT offspring deprived of 6’SL during lactation exhibited consistent alterations in all cognitive functions addressed, hippocampal electrophysiology, and in pathways regulating the serotonergic system (identified through gut microbiota and plasma metabolomics). The impairments observed seem to be associated with a time- (i.e. eye-opening) and region-specific (i.e. pre-frontal cortex) alteration in the expression of genes involved in neurogenesis. In Chapter 2, neonatal access to 3’SL-poor milk resulted in decreased attention, spatial and working memory, and altered LTP compared to the control group Finally, in Chapter 3, I observed similar impairments in cognitive functions compared to the previous studies in mice exposed to reduce neonatal availability of both 3’SL and 6’SL. Regarding the effect of an exogenous administration of 3’SL and 6’SL, the supplementation procedure had reduced the odds to observe the cognitive read-out of the experimental subjects. These results support the hypothesis that early-life dietary sialylated HMOs exert a long-lasting role in the development of cognitive functions.