Differential Susceptibility to Benzo[a]pyrene Exposure during Gestation and Lactation in Mice with Genetic Variations in the Aryl Hydrocarbon Receptor and Cyp1 Genes.

Polycyclic aromatic hydrocarbons are ubiquitous air pollutants, with additional widespread exposure in the diet. PAH exposure has been linked to adverse birth outcomes and long-term neurological consequences. To understand genetic differences that could affect susceptibility following developmental exposure to polycyclic aromatic hydrocarbons, we exposed mice with variations in the aryl hydrocarbon receptor and the three CYP1 enzymes from gestational day 10 (G10) to weaning at postnatal day 25 (P25). We found unexpectedly high neonatal lethality in high-affinity AhrbCyp1b1(-/-) knockout mice compared with all other genotypes. Over 60% of BaP-exposed pups died within their first 5 days of life. There was a significant effect of BaP on growth rates in surviving pups, with lower weights observed from P7 to P21. Again, AhrbCyp1b1(-/-) knockout mice were the most susceptible to growth retardation. Independent of treatment, this line of mice also had impaired development of the surface righting reflex. We used high-resolution mass spectrometry to measure BaP and metabolites in tissues from both dams and pups. We found the highest BaP levels in adipose from poor-affinity AhrdCyp1a2(-/-) dams and identified three major BaP metabolites (BaP-7-OH, BaP-9-OH, and BaP-4,5-diol), but our measurements were limited to a single time point. Future work is needed to understand BaP pharmacokinetics in the contexts of gestation and lactation and how differential metabolism leads to adverse developmental outcomes.

RIPK1 Promotes Energy Sensing by the mTORC1 Pathway.

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

NPC1 Deficiency in Mice is Associated with Fetal Growth Restriction, Neonatal Lethality and Abnormal Lung Pathology.

The rare lysosomal storage disorder Niemann-Pick disease type C1 (NPC1) arises from mutation of NPC1, which encodes a lysosomal transmembrane protein essential for normal transport and trafficking of cholesterol and sphingolipids. NPC1 is highly heterogeneous in both clinical phenotypes and age of onset. Previous studies have reported sub-Mendelian survival rates for mice homozygous for various Npc1 mutant alleles but have not studied the potential mechanisms underlying this phenotype. We performed the first developmental analysis of a Npc1 mouse model, Npc1em1Pav, and discovered significant fetal growth restriction in homozygous mutants beginning at E16.5. Npc1em1Pav/em1Pav mice also exhibited cyanosis, increased respiratory effort, and over 50% lethality at birth. Analysis of neonatal lung tissues revealed lipid accumulation, notable abnormalities in surfactant, and enlarged alveolar macrophages, suggesting that lung abnormalities may be associated with neonatal lethality in Npc1em1Pav/em1Pav mice. The phenotypic severity of the Npc1em1Pav model facilitated this first analysis of perinatal lethality and lung pathology in an NPC1 model organism, and this model may serve as a useful resource for developing treatments for respiratory complications seen in NPC1 patients.

DPP9 enzyme activity controls survival of mouse migratory tongue muscle progenitors and its absence leads to neonatal lethality due to suckling defect.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9ki/ki mice). We show that DPP9ki/ki mice die within 12-18h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

Transgenic rescue of Atg5-null mice from neonatal lethality with neuron-specific expression of ATG5: Systemic analysis of adult Atg5-deficient mice.

Atg5-null mice are neonatal lethal. We have revealed in our recent paper that these mice die due to neuronal dysfunction resulting in suckling failure. Our new mouse model, atg5-/-;Eno2/Nse-Atg5 mice, where Atg5 is deficient in the whole body except for neurons, enables us to analyze the consequences of macroautophagy/autophagy-deficiency in the whole body of adult mice.

Impact of genetic background on neonatal lethality of Gga2 gene-trap mice.

The functional redundancy of the three mammalian Golgi-localized, γ-ear-containing, ADP-ribosylation factor-binding proteins (GGAs) was addressed in a previous study. Using insertional mutagenesis, we found that Gga1 or Gga3 homozygous knockout mice were for the most part normal, whereas mice homozygous for two different Gga2 gene-trap alleles exhibited either embryonic or neonatal lethality in the C57BL/6 background, depending on the source of the vector utilized (Byg vs. Tigm, respectively). We now show that the Byg strain harbors a disrupted Gga2 allele that is hypomorphic, indicating that the Byg lethality is attributable to a mechanism independent of GGA2. This is in contrast to the Tigm Gga2 allele, which is a true knockout and establishes a role for GGA2 during the neonatal period. Placement of the Tigm Gga2 allele into the C57BL6/Ola129Sv mixed background results in a lower incidence of neonatal lethality, showing the importance of genetic background in determining the requirement for GGA2 during this period. The Gga2(-/-) mice that survive have reduced body weight at birth and this runted phenotype is maintained through adulthood.