Probes for INS

During the Zika epidemic in Brazil, a baby was born at term with microcephaly and arthrogryposis. The mother had Zika symptoms at 10 weeks of gestation. At 17 weeks, ultrasound showed cerebral malformation and ventriculomegaly. At 24 weeks, the amniotic fluid contained ZIKV RNA and at birth, placenta and maternal blood were also positive using RT-qPCR. At birth the baby urine contained ZIKV RNA, whereas CSF at birth and urine at 17 days did not. Seizures started at 6 days. EEG was abnormal and CT scan showed cerebral atrophy, calcifications, lissencephaly, ventriculomegaly, and cerebellar hypoplasia. Bacterial sepsis at 2 months was treated. A sudden increase in head circumference occurred at 4 months necessitating ventricle-peritoneal shunt placement. At 5 months, the infant died with sepsis due to bacterial meningitis. Neuropathological findings were as severe as some of those found in neonates who died soon after birth, including hydrocephalus, destructive lesions/calcification, gliosis, abnormal neuronal migration, dysmaturation of nerve cells, hypomyelination, loss of descending axons, and spinal motor neurons. ZIKV RNA was detected only in frozen brain tissue using RT-qPCR, but infected cells were not detected by in situ hybridization. Progressive gliosis and microgliosis in the midbrain may have contributed to aqueduct compression and subsequent hydrocephalus. The etiology of progressive disease after in utero infection is not clear and requires investigation.

Abstract

BACKGROUND:

Vertical transmission of Zika virus (ZIKV) leads to infection of neuroprogenitor cells and destruction of brain parenchyma. Recent evidence suggests that the timing of infection as well as host factors may affect vertical transmission. As a result, congenital ZIKV infection may only become clinically apparent in the postnatal period.

OBJECTIVES:

We sought to develop an outbred mouse model of ZIKV vertical transmission to determine if the timing of gestational ZIKV exposure yields phenotypic differences at birth and through adolescence. We hypothesized that later gestational inoculations would only become apparent in adolescence.

METHODS:

To better recapitulate human exposures, timed pregnant Swiss-Webster dams (n=15) were subcutaneously inoculated with 1x104PFU of first passage contemporary ZIKV HN16 strain or a mock injection on embryonic day 4, 8, or 12 with bioactive anti-interferon alpha receptor antibody administered in days preceding and proceeding inoculation. The antibody was given to prevent the robust type I interferon signaling cascade that make mice inherently resistant to ZIKV infection. At birth and adolescence (6 weeks of age) offspring were assessed for growth, brain weight and biparietal head diameters (BPD), and ZIKV viral levels by RT-PCR or in situ hybridization.

RESULTS:

Pups of ZIKV-infected dams infected at e4 and e8 but not e12 were growth restricted (p<0.003). Brain weights were significantly smaller at birth (p=0.01) for e8 ZIKV-exposed offspring. At 6 weeks of age, biparietal diameters (BPD) were smaller for all ZIKV exposed males and females (p<0.05), with e8 exposed males smallest by BPD and growth restriction measurements (weight >2 SD, p=0.0007). All pups and adolescent mice were assessed for ZIKV infection by RT-PCR. Analysis of all underweight pups reveled one to be positive for neuronal ZIKV infection by in situ hybridization, while a second moribund animal was diffusely positive at 8 days of age by ZIKV infectivity throughout the brain, kidneys and intestine.

CONCLUSION:

These findings demonstrate that postnatal effects of infection occurring at single time points continue to be detrimental to offspring in the postnatal period in a subset of littermates and subject to a window of gestational susceptibility coinciding with placentation. This model recapitulates frequently encountered clinical scenarios in non-endemic regions, including the majority of the U.S., where travel related exposure occurs in short and well-defined windows of gestation. Our low rate of infection and relatively rare evidence of congenital Zika syndrome parallels human population-based data.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

In the arcuate nucleus of the hypothalamus (ARH) satiety signaling (anorexigenic) pro-opiomelanocortin (POMC)-expressing and hunger signaling (orexigenic) agouti-related peptide (AgRP)-expressing neurons are key components of the neuronal circuits that control food intake and energy homeostasis. Here, we assessed whether the catecholamine noradrenalin directly modulates the activity of these neurons in mice. Perforated patch clamp recordings showed that noradrenalin changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the opposing effect on these neurons is mediated by the activation of excitatory α1A - and β- adrenergic receptors in NPY/AgRP neurons, while POMC neurons are inhibited via α2A - adrenergic receptors. Thus, the coordinated differential modulation of the key hypothalamic neurons in control of energy homeostasis assigns noradrenalin an important role to promote feeding.

The development of interventions to prevent congenital Zika syndrome (CZS) has been limited by the lack of an established nonhuman primate model. Here we show that infection of female rhesus monkeys early in pregnancy with Zika virus (ZIKV) recapitulates many features of CZS in humans. We infected 9 pregnant monkeys with ZIKV, 6 early in pregnancy (weeks 6-7 of gestation) and 3 later in pregnancy (weeks 12-14 of gestation), and compared findings with uninfected controls. 100% (6 of 6) of monkeys infected early in pregnancy exhibited prolonged maternal viremia and fetal neuropathology, including fetal loss, smaller brain size, and histopathologic brain lesions, including microcalcifications, hemorrhage, necrosis, vasculitis, gliosis, and apoptosis of neuroprogenitor cells. High-resolution MRI demonstrated concordant lesions indicative of deep gray matter injury. We also observed spinal, ocular, and neuromuscular pathology. Our data show that vascular compromise and neuroprogenitor cell dysfunction are hallmarks of CZS pathogenesis, suggesting novel strategies to prevent and to treat this disease.

Neurotensin, a gut hormone and neuropeptide, increases in circulation after bariatric surgery in rodents and humans and inhibits food intake in mice. However, its potential to treat obesity and the subsequent metabolic dysfunctions have been difficult to assess owing to its short half-life in vivo Here, we demonstrate that a long acting, pegylated analogue of the neurotensin peptide (P-NT) reduces food intake, body weight and adiposity in diet-induced obese (DIO) mice when administered once daily for 6 days. Strikingly, when P-NT was combined with the GLP-1 mimetic liraglutide the two peptides synergized to reduce food intake and body weight relative to each mono-therapy, without inducing a taste aversion. Further, P-NT and liraglutide co-administration improved glycemia and reduced steatohepatitis. Finally, we show that the melanocortin pathway is central for P-NT-induced anorexia and necessary for the full synergistic effect of P-NT and liraglutide combination-therapy. Overall, our data suggest that P-NT and liraglutide combination-therapy could be an enhanced treatment for obesity with improved tolerability compared to liraglutide mono-therapy.

Excess caloric intake results in increased fat accumulation and an increase in energy expenditure via diet-induced adaptive thermogenesis; however, the underlying mechanisms controlling these processes are unclear. Here we identify the neuropeptide FF receptor-2 (NPFFR2) as a critical regulator of diet-induced thermogenesis and bone homoeostasis. Npffr2-/- mice exhibit a stronger bone phenotype and when fed a HFD display exacerbated obesity associated with a failure in activating brown adipose tissue (BAT) thermogenic response to energy excess, whereas the activation of cold-induced BAT thermogenesis is unaffected. NPFFR2 signalling is required to maintain basal arcuate nucleus NPY mRNA expression. Lack of NPFFR2 signalling leads to a decrease in BAT thermogenesis under HFD conditions with significantly lower UCP-1 and PGC-1α levels in the BAT. Together, these data demonstrate that NPFFR2 signalling promotes diet-induced thermogenesis via a novel hypothalamic NPY-dependent circuitry thereby coupling energy homoeostasis with energy partitioning to adipose and bone tissue.