Leo Enthoven

Hypomethylation of D4Z4 in 4q-linked and non-4q-linked facioscapulohumeral muscular dystrophy

The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD1, OMIM 158900) is caused by contraction of the D4Z4 repeat array on 4qter. We show that this contraction causes marked hypomethylation of the contracted D4Z4 allele in individuals with FSHD1. Individuals with phenotypic FSHD1, who are clinically identical to FSHD1 but have an unaltered D4Z4, also have hypomethylation of D4Z4. These results strongly suggest that hypomethylation of D4Z4 is a key event in the cascade of epigenetic events causing FSHD1.

The postnatal development of the hypothalamic-pituitary-adrenal axis in the mouse.

The main characteristic of the postnatal development of the stress system in the rat is the so‐called stress hypo‐responsive period (SHRP). Lasting from postnatal day (pnd) 4 to pnd 14, this period is characterized by very low basal corticosterone levels and an inability of mild stressors to induce an enhanced ACTH and corticosterone release. During the last years, the mouse has become a generally used animal in stress research, also due to the wide availability of genetically modified mouse strains. However, very few data are available on the ontogeny of the stress system in the mouse. This study therefore describes the postnatal ontogeny of peripheral and central aspects of the hypothalamic–pituitary–adrenal (HPA) axis in the mouse. We measured ACTH and corticosterone in blood and CRH, urocortin 3 (UCN3), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) transcripts in the brain at postnatal days 1, 2, 4, 6, 9, 12, 14 and 16. Our results show that we can subdivide the postnatal development of the HPA axis in the mouse in two phases. The first phase corresponds to the SHRP in the rat and lasts from right after birth (pnd 1) until pnd 12. Basal corticosterone levels were low and novelty exposure did not enhance corticosterone or ACTH levels. This period is further characterized by a high expression of CRH in the paraventricular nucleus (PVN) of the hypothalamus. Expression levels of GR in the hippocampus and UCN3 in the perifornical area are low at birth but increase significantly during the SHRP, both reaching the highest expression level at pnd 12. In the second phase, the mice have developed past the SHRP and were now exhibiting enhanced corticosterone basal levels and a response of ACTH and corticosterone to mild novelty stress. CRH expression was decreased significantly, while expression of UCN3 and GR remained high, with a small decrease at pnd 16. The expression of MR in the hippocampus was very dynamic throughout the postnatal development of the HPA axis and changed in a time and subregion specific manner. These results demonstrate for the first time the correlation between the postnatal endocrine development of the mouse and gene expression changes of central regulators of HPA axis function.

The dynamics of the hypothalamic-pituitary-adrenal axis during maternal deprivation.

A close contact between the dam and the litter is essential for the normal development of the hypothalamic‐pituitary‐adrenal (HPA) axis in rats and mice. Maternal signals, as licking and feeding, have been shown to sustain the HPA axis of the pups in a hypo‐responsive state. Disruption of this mother–pup interaction by 24 h of maternal deprivation activates the otherwise quiescent stress system of the neonates, resulting in an enhanced adrenal sensitivity to adrenocorticotropic hormone (ACTH) and a decreased expression of central HPA markers, such as corticotropin‐releasing hormone (CRH). However, the dynamics of these central and peripheral changes over the 24h period are largely unknown. In this study, we examined the time course of some of the central and peripheral indices of HPA activity during 24 h of maternal deprivation. We measured corticosterone and ACTH in the blood as well as CRH, mineralocorticoid and glucocorticoid receptor expression in the brain. Our results demonstrate that each of the components of the HPA axis responds to maternal deprivation at different time points following removal of the mother and with a very specific time course. The main activation of the HPA axis occurred between 4 h and 8 h of maternal absence. By contrast, during the second half of the deprivation period, negativefeedback mechanisms restrained the further increase in ACTH and corticosterone release. We conclude that maternal deprivation triggers a cascade of sequential changes at the various levels of the stress system, and that measuring only one aspect of the system at one time point does not accurately reflect the dynamic alterations of the HPA axis.

Variable hypomethylation of D4Z4 in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) progressively affects the facial, shoulder, and upper arm muscles and is associated with contractions of the polymorphic D4Z4 repeat array in 4q35. Recently, we demonstrated that FSHD alleles are hypomethylated at D4Z4. To study potential relationships between D4Z4 hypomethylation and both residual repeat size and clinical severity, we compared the clinical severity score with D4Z4 methylation in unrelated FSHD patients. Correcting the clinical severity score for age at examination improves the parameter to define clinical severity and provides further support for hypomethylation of FSHD alleles. However, a linear relationship between repeat size and clinical severity of the disease cannot be established. Interestingly, FSHD can be separated in two clinical severity classes: patients with residual repeat sizes of 10 to 20kb are severely affected and show pronounced D4Z4 hypomethylation. In contrast, patients with repeat sizes of 20 to 31kb show large interindividual variation in clinical severity and D4Z4 hypomethylation. Because the majority of familial FSHD cases are represented in this interval and considering the overt variation in clinical severity in these familial cases, it thus is imperative to develop comprehensive allele‐specific assays monitoring total D4Z4 methylation to investigate whether interindividual variation in D4Z4 methylation can be translated into a prognostic factor for clinical severity. Ann Neurol 2005;58:569–576

Age-Related Changes in Hypothalamic-Pituitary-Adrenal Axis Activity of Male C57BL/6J Mice

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.

Hypothalamic-Pituitary-Adrenal Axis Activity of Newborn Mice Rapidly Desensitizes to Repeated Maternal Absence but Becomes Highly Responsive to Novelty

In CD1 mice we investigated the hypothalamic-pituitary-adrenal (HPA) axis response to maternal separation for 8 h daily from postnatal d 3 to 5. At d 3 a slow separation-induced corticosterone response developed that peaked after 8 h, and the pups became responsive to stressors. On the second and third day, the response to 8 h separation rapidly attenuated, whereas the response to novelty did not, a pattern reflected by the hypothalamic c-fos mRNA response. If maternal separation and exposure to novelty were combined, then after the third such daily exposure, the sensitivity to the stressor was further enhanced. Meanwhile, basal corticosterone and ACTH levels were persistently suppressed 16 h after pups were reunited with their mothers. To explain the HPA axis desensitization after repeated separation, we found that circulating ghrelin levels increased and glucose levels decreased after all periods of maternal separation, ruling out a role of altered metabolism. Glucocorticoid feedback was not involved either because a glucocorticoid receptor antagonist amplified the corticosterone response after the first but became ineffective after the third separation. In contrast, a mineralocorticoid receptor antagonist decreased and increased corticosterone levels after the first and third period of separation, respectively. In conclusion, the newborns HPA axis readily desensitizes to repeated daily maternal separation, but continues to respond to novelty in a manner influenced by a central mineralocorticoid receptor- rather than glucocorticoid receptor-mediated mechanism.

Differential development of stress system (re)activity at weaning dependent on time of disruption of maternal care

Maternal deprivation, a separation of mother and pups for 24 h in the first weeks of life has long-lasting consequences for the glucocorticoid stress system in rats. We examined in male CD1 mice whether the postnatal day (pnd) of deprivation determines the (re)activity of the stress system at weaning under basal and novelty stress conditions. Maternal deprivation was only effective when applied within the stress hypo-responsive period (SHRP) between pnds 1 and 12, but not on pnd 13. Maternal deprivation (i) early in the SHRP (pnd 3) resulted in lower hippocampal GR mRNA expression together with a prolonged corticosterone response to stress; while (ii) late in the SHRP (pnd 8) the amplitude of the ACTH response to stress was enhanced. (iii) Strikingly, the effects of the double deprivation (pnds 3 and 8) were not additive: sustained, stress non-responsive high plasma ACTH concentrations with corticosterone indistinguishable from control animals coincided with a lower expression of hippocampal MR and GR mRNA. These results present species-specific effects (mouse versus rat) of an adverse early life event on HPA axis regulation at weaning. A subsequent deprivation experience interferes with the effects of earlier deprivation. We conclude that the developmental stage of the organism determines the vulnerability for the detrimental effects of maternal deprivation and the organization of the stress system in adolescence.

Increased corticosterone secretion and early-onset of cognitive decline in female apolipoprotein E-knockout mice

In the present study, the interaction of age and apolipoprotein E (apoE)-genetic background on cognitive abilities was investigated in young (5-6 months) and aged (14-16 months) female apolipoprotein E-knockout (apoE0/0) and wild-type mice. Cognitive abilities are known to be affected by the steroid hormones corticosterone and estrogen. Therefore, we measured the activity and reactivity of the hypothalamic-pituitary-adrenal (HPA) axis expressed by circadian corticosterone concentrations and responses to novelty and controlled the regularity of the estrous cycle. Young female apoE0/0 mice acquired the water maze task and showed a similar latency and search strategy to locate the platform as young female wild-type mice. Similar corticosterone responses to novelty were observed in both genotypes. Regularity of the estrous cycle was disturbed in a small percentage of the young apoE0/0 female mice. However, in aged female apoE0/0 mice water maze performance was impaired with search strategies less persistent than in aged wild-type mice. In parallel, increased corticosterone concentrations were measured in apoE0/0 mice in response to novelty and during the circadian cycle. The percentage of mice with an irregular estrous cycle increased with age, but was comparable for apoE0/0 and wild-type mice. Thus, although disruption of the apoE gene affects the regularity of the estrous cycle in young mice, it is the enhanced corticosterone secretion, which parallels the cognitive decline in the aging female apoE0/0 mice.

Effects of maternal deprivation of CD1 mice on performance in the water maze and swim stress.

Rat pups subjected to a single 24h maternal deprivation show altered stress responsiveness and cognitive performance in the water maze in adulthood. Here we show in 6-month-old male CD1 mice (deprived 24h at postnatal day 8) an initial impairment in reversal learning: relocating the platform revealed perseverance in search for the former location. Spatial learning, long-term memory and swim-induced corticosterone responses were not affected. We conclude that reduced flexibility is a subtle long-lasting behavioural change induced by maternal deprivation.

Ontogeny of the HPA axis of the CD1 mouse following 24 h maternal deprivation at pnd 3.

One of the striking characteristics of the developing neuroendocrine system of rats and mice is the stress hypo‐responsive period (SHRP), i.e. low basal corticosterone secretion and the inability to increase corticosterone in response to mild stressors during the first 2 weeks of life. However, immediately after 24 h of deprivation from maternal care the response of the hypothalamic–pituitary–adrenal (HPA) axis to mild stressors is enhanced. This study examines in CD1 mouse pups the recovery pattern of markers of HPA axis (re)activity from maternal deprivation (once for 24 h from postnatal days (pnds) 3 to 4). As expected, deprivation induced a profound corticosterone response to novelty immediately after deprivation. In contrast, 1 day after reunion with the mother (pnd 5), this effect was abolished, lasting for at least 3 days. Basal corticosterone remained even below control levels. Corticotropin‐releasing hormone (CRH) mRNA expression in the hypothalamic paraventricular nucleus (PVN) was suppressed for 2 days, exceeded control levels at pnds 7 and 8, and subsequently followed the gradual decline observed in controls until pnd 12. Delayed and rather short‐lasting changes were found for adrenocorticotropic hormone (low at pnd 5), and glucocorticoid receptor mRNA expression (decreased in the PVN at pnd 4, and in the hippocampal CA1 area at pnd 5). Hippocampal mineralocorticoid receptor mRNA expression was unaffected. From pnds 9 to 13, both deprived and control pups gradually emerged from the SHRP in a similar temporal pattern. In conclusion, maternal deprivation at pnd 3 augments hypo‐responsiveness of corticosterone secretion to mild stress for several days, but does not affect the duration of the SHRP. Whether CRH and glucocorticoid receptor changes are cause or consequence remains to be established.