Terry Gemelli

Postnatal Loss of Methyl-CpG Binding Protein 2 in the Forebrain is Sufficient to Mediate Behavioral Aspects of Rett Syndrome in Mice

BACKGROUND Mutations in the methyl-CpG binding protein 2 (MeCP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder that is accompanied by a broad array of behavioral phenotypes, mainly affecting females. Methyl-CpG binding protein 2 is a transcriptional repressor that is widely expressed in all tissues. METHODS To investigate whether the postnatal loss of MeCP2 in the forebrain is sufficient to produce the behavioral phenotypes observed in RTT, we have generated conditional MeCP2 knockout mice. RESULTS These mice display behavioral abnormalities similar to RTT phenotypes, including hindlimb clasping, impaired motor coordination, increased anxiety, and abnormal social behavior with other mice. These mice, however, have normal locomotor activity and unimpaired context-dependent fear conditioning, suggesting that the behavioral deficits observed are the result of loss of MeCP2 function in postnatal forebrain and not the result of generalized global deficits. CONCLUSIONS These data highlight the important role of MeCP2 in the forebrain and suggest that even partial loss of MeCP2 expression in these brain regions is sufficient to recapitulate features of RTT.

Oxytocin Deficiency Mediates Hyperphagic Obesity of Sim1 Haploinsufficient Mice

Single-minded 1 (Sim1) encodes a transcription factor essential for formation of the hypothalamic paraventricular nucleus (PVN). Sim1 haploinsufficiency is associated with hyperphagic obesity and increased linear growth in humans and mice, similar to the phenotype of melanocortin 4 receptor (Mc4r) mutations. PVN neurons in Sim1(+/-) mice are hyporesponsive to the melanocortin agonist melanotan II. PVN neuropeptides oxytocin (Oxt), TRH and CRH inhibit feeding when administered centrally. Consequently, we hypothesized that altered PVN neuropeptide expression mediates the hyperphagia of Sim1(+/-) mice. To test this hypothesis, we measured hypothalamic expression of PVN neuropeptides in Sim1(+/-) and wild-type mice. Oxt mRNA and peptide were decreased by 80% in Sim1(+/-) mice, whereas TRH, CRH, arginine vasopressin (Avp), and somatostatin mRNAs were decreased by 20-40%. Sim1(+/-) mice also showed abnormal regulation of Oxt but not CRH mRNA in response to feeding state. A selective Mc4r agonist activated PVN Oxt neurons in wild-type mice, supporting involvement of these neurons in melanocortin feeding circuits. To test whether Oxt itself regulates feeding, we measured the effects of central administration of an Oxt receptor antagonist or repeated doses of Oxt on food intake of Sim1(+/-) and wild-type mice. Sim1(+/-) mice were hypersensitive to the orexigenic effect of the Oxt receptor antagonist. Oxt decreased the food intake and weight gain of Sim1(+/-) mice at a dose that did not affect wild-type mice. Our results support the importance of Oxt neurons in feeding regulation and suggest that reduced Oxt neuropeptide is one mechanism mediating the hyperphagic obesity of Sim1(+/-) mice.

Postnatal Sim1 Deficiency Causes Hyperphagic Obesity and Reduced Mc4r and Oxytocin Expression

Single-minded 1 (SIM1) mutations are one of the few known causes of nonsyndromic monogenic obesity in both humans and mice. Although the role of Sim1 in the formation of the hypothalamus has been described, its postdevelopmental, physiological functions have not been well established. Here we demonstrate that postnatal CNS deficiency of Sim1 is sufficient to cause hyperphagic obesity. We conditionally deleted Sim1 after birth using CaMKII-Cre (α-calcium/calmodulin-dependent protein kinase II-Cre) lines to recombine a floxed Sim1 allele. Conditional Sim1 heterozygotes phenocopied germ line Sim1 heterozygotes, displaying hyperphagic obesity and increased length. We also generated viable conditional Sim1 homozygotes, demonstrating that adult Sim1 expression is not essential for mouse or neuron survival and revealing a dosage-dependent effect of Sim1 on obesity. Using stereological cell counting, we showed that the phenotype of both germ line heterozygotes and conditional Sim1 homozygotes was not attributable to global hypocellularity of the paraventricular nucleus (PVN) of the hypothalamus. We also used retrograde tract tracing to demonstrate that the PVN of germ line heterozygous mice projects normally to the dorsal vagal complex and the median eminence. Finally, we showed that conditional Sim1 homozygotes and germ line Sim1 heterozygotes exhibit a remarkable decrease in hypothalamic oxytocin (Oxt) and PVN melanocortin 4 receptor (Mc4r) mRNA. These results demonstrate that the role of Sim1 in feeding regulation is not limited to formation of the PVN or its projections and that the hyperphagic obesity in Sim1-deficient mice may be attributable to changes in the leptin–melanocortin–oxytocin pathway.

DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

Inducible neuronal inactivation of Sim1 in adult mice causes hyperphagic obesity.

Germline haploinsufficiency of human or mouse Sim1 is associated with hyperphagic obesity. Sim1 encodes a transcription factor required for proper formation of the paraventricular (PVN), supraoptic, and anterior periventricular hypothalamic nuclei. Sim1 expression persists in these neurons in adult mice, raising the question of whether it plays a physiologic role in regulation of energy balance. We previously showed that Sim1 heterozygous mice had normal numbers of PVN neurons that were hyporesponsive to melanocortin 4 receptor agonism and showed reduced oxytocin expression. Furthermore, conditional postnatal neuronal inactivation of Sim1 also caused hyperphagic obesity and decreased hypothalamic oxytocin expression. PVN projections to the hindbrain, where oxytocin is thought to act to modulate satiety, were anatomically intact in both Sim1 heterozygous and conditional knockout mice. These experiments provided evidence that Sim1 functions in energy balance apart from its role in hypothalamic development but did not rule out effects of Sim1 deficiency on postnatal hypothalamic maturation. To address this possibility, we used a tamoxifen-inducible, neural-specific Cre transgene to conditionally inactivate Sim1 in adult mice with mature hypothalamic circuitry. Induced Sim1 inactivation caused increased food and water intake and decreased expression of PVN neuropeptides, especially oxytocin and vasopressin, with no change in energy expenditure. Sim1 expression was not required for survival of PVN neurons. The results corroborate previous evidence that Sim1 acts physiologically as well as developmentally to regulate body weight. Inducible knockout mice provide a system for studying Sim1s physiologic function in energy balance and identifying its relevant transcriptional targets in the hypothalamus.

Evolution of the skin manifestations of X-linked pigmentary reticulate disorder

DEAR EDITOR, X-linked reticulate pigmentary disorder (XLPDR, MIM 301220) is a rare syndrome first recognized by Partington. The cardinal manifestations of the disorder are diffuse reticulate hyperpigmentation, hypohidrosis and unique facial features, as well as recurrent pneumonias and sterile inflammation in various organs. The syndrome is exceedingly rare and only 20 patients have been reported worldwide. Recently, we identified that the disorder is associated with a recurrent intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase alpha. Here, we report a new case of XLPDR and include the first detailed description of the evolution of its dermatological features, as well as confirmation of the same intronic POLA1 mutation. The patient is the fourth child of four and has an unaffected elder sister (Fig. S1a; see Supporting Information). His elder brother has an atrial septal defect, but no other abnormalities. Another brother was born with Potter syndrome and died at 2 h of age. The patient’s mother has restricted hyperpigmentation along Blaschko lines (Fig. S1b; see Supporting Information), which is also observed in his grandmother and aunt, but no other males in the family appear clinically affected. During his first year of life, the patient developed recurrent pneumonia, urethral strictures with occasional bleeding after urination, and bloody diarrhoea (which eventually resolved while on hydrolyzed formula). However, he had no recurrent or unusual nonpulmonary infections and did not have growth delay. At age 7 years, he developed bronchiectasis in the left lower lobe (requiring pulmozyme treatment) and was noted to have decreased visual acuity and photophobia due to bilateral erosive keratitis. At age 13 years, the typical facial features of XLPDR (upswept hair and flared eyebrows) along with reticulate hyperpigmentation were noted (Fig. S1c; see Supporting Information). Laboratory studies (Table S1; see Supporting Information) were otherwise within normal ranges. The diagnosis of XLPDR was confirmed at age 13 years by genetic and immunological tests. The patient was found to be hemizygous for a previously reported POLA1 mutation (NC_000023.10:g.24744696A>G). This noncoding mutation in intron 13 of the POLA1 gene is known to be associated with activated type I interferon responses, and indeed the patient displayed dramatic elevation of interferon-stimulated genes (Fig. S1d; see Supporting Information). While it has been recognized that the pigmentary changes in XLPDR are not present at birth, the evolution of the skin phenotype has not been reported. Retrospective analysis of the new proband indicates no overt changes in skin pigmentation in the first year of life (Fig. S1e; see Supporting Information). The first dermatological manifestations were noted at 14 months of age, when he developed facial telangiectasias (Fig. S1f; see Supporting Information). At the same age, the patient developed hypopigmented spots on the face, extremities and ultimately the trunk (Fig. S1g; see Supporting Information). These skin changes were not associated with pruritus or pain, but the skin was persistently dry. Frank hypohidrosis was noted in infancy, selectively affecting trunk and extremities. In contrast, the head and neck presented profuse sweating since this age. In view of these changes, at age 7 years, a skin biopsy was obtained from his left thigh, which revealed epidermal hypomelanosis with orthokeratosis, focal acanthosis, focal lymphocytosis and mild hypopigmentation (Fig. S1h; see Supporting Information). The dermis contained few melanophages and minimal superficial lymphocytic perivascular infiltrate. Direct immunofluorescence revealed granular deposits of C3 at the dermoepidermal junction, but no deposition of IgG, IgA, IgM or fibrin. Hyperpigmentation was first noted by the parents at 8 years old. At 12 years old, a new skin biopsy revealed orthokeratosis with areas of hyperpigmentation and hypopigmentation of the basal layer of the epidermis. The dermis had mild perivascular inflammatory infiltrates composed of lymphocytes and some melanophages (Fig. S1i; see Supporting Information). Our findings in this case further confirm that XLPDR is associated with constitutive activation of type I interferon responses. Moreover, these data demonstrate lack of genetic heterogeneity in XLPDR, meaning that the identical mutation has been seen in all cases thus far. We speculate that the unique features of the syndrome may be directly related to the specific effects induced by this intronic variant, such as possible variability in mis-splicing among tissues. Unlike Aicardi–Gouti eres syndrome (AGS), the best known interferonopathy, XLPDR does not involve the brain and is associated with a unique set of manifestations including the characteristic facial features and hypohidrosis, which remain unexplained. Among type I interferonopathies, skin findings are seen in activating mutations in TMEM173 (STING), which lead to pulmonary and skin vasculitis, predominantly affecting digits. Additionally, ADAR mutations, which have been linked to AGS, can also result in dyschromatosis symmetrica hereditaria (MIM 127400), characterized by the development in early childhood