English summary thesis Maralinde Abbink
Early-life is a critical period for brain development, during which the brain is shaped for life. This ‘programming’ of the brain can be negatively influenced by adversity in the early-life environment, such as abuse and neglect. Early-life adversity can indeed lastingly impact brain structure and function and thereby increase the risk for the development of psychopathologies and cognitive deficits later in life. In recent years, this developmental origin of health and disease (DOHaD) hypothesis has received quite some attention in the context of early-life stress (ES) and extensive literature has shown that ES can induce cognitive dysfunction and alter disease susceptibility in later life. However, the contribution of lifestyle factors and environ- mental exposure to this ES-enhanced predisposition for psychopathology has only received marginal attention so far.

Sadly, ES is a global problem that is difficult to prevent. As currently no effective treatments are available, it becomes increasingly important to understand the contribution of adult lifestyle on the increased risk for psychopathology following early-life exposure to stress. Increasing our knowledge on this topic will help to identify vulnerable populations and might contribute to establishing (preventive) therapeutic strategies to improve adult health outcome after ES. Unraveling the underlying biological mechanisms involved in this process is therefore essential. In this thesis, we aimed to gain further insight in the mechanisms underlying ES programming and the contribution of lifestyle or environmental influences to the increased susceptibility to cognitive impairment as a consequence of ES. We here focused on astrocytes and neurogenesis in the hippocampus.

In chapter 1, we reviewed the existing literature on the effects of early-life adversity on astrocytes. We show that different types of early-life adversity (i.e. maternal depri- vation/separation, immune challenges, and malnutrition), impact astrocytes both on the short- and long-term. The main findings implicate alterations in astrocyte glial fibrillary acidic protein (GFAP) expression per se as well as persistent alterations in astrocyte metabolism related factors, like glutamate and glucose transporters. In addition, we propose that astrocytes may be key players in the integration of a variety of signals from the early environment, thereby contributing to the synergistic effect of hormonal, inflammatory and nutritional input from the adverse early-life environment on the brain. Lastly, we highlight the possibility of using human-based iPSC models to increase our knowledge on astrocyte biology and the interaction of astrocytes with their environment in the context of early-life adversity.

We extended our knowledge on the lifelong effects of ES on astrocytes and studied whether ES modulates the response of astrocytes to amyloid pathology in a transgenic mouse model of Alzheimer’s disease (AD) in chapter 2. We showed that ES, induced by housing mice litters with limited nesting and bedding material, affects hippocampal GFAP expression in an age-dependent manner. In addition, we found that APP/PS1 overexpression does not result in any astrocytic changes at an early-pathological stage of 4 months, but affects GFAP expression in both the hippocampus and entorhinal cortex in different directions and alters hippocampal gene expression of astrocyte-related genes at a late-pathological stage of 10 months. Interestingly, ES did not modulate the effects of amyloid pathology on astrocytic features, unlike microglia, that were previously shown to have an altered neuroinflammatory response to amyloid pathology in mice that were exposed to ES. However, we show that locally clustered GFAP expression only present in the hippocampus of APP/PS1 mice, correlates to CD68 expression, which indicates a relation between phagocytic microglia and astroglial GFAP in the context of amyloid pathology. These results highlight that the response of astrocytes to AD pathology depends on plaque association, brain region, and disease progression, but is not modulated by early-life exposure to stress.

To further study ES programming of astrocytes, we examined ES-induced priming of the astrocyte response to stress hormones in chapter 3. In this preliminary report, we used a combined in vitro and in vivo approach in which primary hippocampal astrocyte cultures were derived from ES mice and subsequently treated with the synthetic glucocorticoid dexamethasone (DEX). We show that DEX significantly alters gene expression levels of astrocyte-related genes, an effect that was modulated by ES for certain genes. This preliminary report provides initial evidence on ES-induced priming of the astrocyte response to stress hormones.

In chapter 4, we applied nutritional interventions in order to combat ES-induced effects on the brain. We replicated previous results by demonstrating that an early-life diet with an improved ω-6/ω-3 fatty acid ratio, rescued cognitive impairments induced by previous exposure to ES. In addition, we show that both ES and a low ω-6/ω-3 dietary ratio during early-life alters the peripheral and central response to an immune challenge in adulthood (i.e. LPS): i) ES exacerbated the LPS-induced increase in plasma cytokine levels and ii) both ES and early-diet modulated hippocampal gene expression changes in response to LPS, as measured by microarray. Interestingly, no changes in plasma cytokine levels or hippocampal gene expres- sion were observed under basal conditions (i.e. no LPS), highlighting that ES alters the responsiveness to later life environmental factors without impairments being apparent under normal conditions. These initial results already provide novel evidence on the programming of both the peripheral and central immune responses by both stress and dietary exposures during early-life. In this ongoing study, we will later investigate the biological pathways involved. The genome-wide approach of this study allows identification of possible new underlying mechanisms involved in ES-induced effects on the brain.

To determine the effect of ES and later lifestyle factors at the level of neurogenesis, i.e. the birth of new neurons from stem cells in the adult brain, we started out in chapter 5 to investigate the effect of ES on the neural stem progenitor cell (NSPC) pool in the hippocampus. Using a transgenic mouse line for Nestin-GFP, a commonly used NSPC reporter, we found that ES does not deplete the adult NSPC pool in 4-month-old mice. Our findings indicate that the reduction in adult hippocampal neurogenesis in adulthood generally observed after previous ES exposure is likely not the direct consequence of a depleted NSPC pool.

In chapter 6, we continued our research on effects of ES in the context of early nutritional interventions in combination with a negative challenge in adulthood: an ‘unhealthy’ Western-style diet (WSD). We studied whether postnatal exposure to infant milk formula (IMF) altered in structure to mimic the physical characteristics of breast milk (Nuturis®), modulates the effects of ES on brain and metabolic outcomes in response to an unhealthy WSD in adulthood. Focusing on both mental and metabolic parameters is important as ES is not only a major risk factor for psychopathologies, but also increases the risk to develop metabolic disorders. Notably, psychiatric and metabolic disorders are often highly comorbid. We demonstrated for the first time that a short-term exposure to an IMF physically mimicking breast milk has a lasting impact on adult hippocampal neurogenesis in response to an unhealthy diet in adulthood. Metabolically, ES and Nuturis® had limited effects on WSD-induced body composition changes, but patterns were identified for i) ES-enhanced susceptibility to the negative effects of WSD on metabolic factors, and ii) an attenuation of this effect by postnatal exposure to Nuturis®. However, lifelong WSD exposure abolished these effects, which stresses the substantial negative impact of a long-lasting unhealthy diet, a clinically relevant problem in current society.

In chapter 7 we further studied the impact of adult lifestyle factors on ES effects. Here, in contrast to the negative adult lifestyle factor studied in chapter 6, we focused on exercise, a well-known pro-neurogenic stimulus. Strikingly, we show that female mice that were previously exposed to ES lacked the increase in neurogenesis in response to running, while no baseline reductions in neurogenesis were present as observed in male mice. This; i) further supports the notion that functional deficits as a consequence of ES may only become apparent under the influence of later life environmental challenges or exposures, and ii) highlights the importance of study- ing both male and female subjects in the context of early-life adversity, as ES-in- duced alterations appear sex-specific. We have for the first time provided evidence for an ES-induced unresponsiveness to exercise in adult/middle-aged female mice.

Finally, in chapter 8, the main findings of this thesis are summarized and discussed from a broader perspective. First, the role of astrocytes in early-life adversity induced programming of the brain is discussed. I discuss the importance of further studying the involvement of astrocytes in the context of ES, as astrocytes are increasingly implicated in stress-related disorders (e.g. depression) and diseases for which ES is a known risk factor (e.g. AD). In this context, it is crucial to further investigate astrocyte heterogeneity for which single cell analysis tools are available. Secondly, I discuss the potential of lifestyle (i.e. nutritional and exercise-based) interventions to protect against the negative effects of ES on the brain. In this context, I focused on hippocampal plasticity and specifically adult hippocampal neurogenesis, and speculate that immune and neurogenic processes may underlie the beneficial effect of increased ω-3 availability on cognitive functioning in ES mice. In addition, the implications of the altered lipid structure in Nuturis® on neurodevelopment are discussed. Lastly, we discuss the modulation of ES-induced effects by lifestyle and environmental factors and discuss the implications for specific therapeutic interven- tions and time-windows, and their clinical relevance.