GM2 gangliosidoses, including Tay-Sachs disease (TSD) and Sandhoff disease (SD), are lysosomal storage disorders caused by deficiencies in β-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system dysfunction (CNS). Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or β-subunits at a 1:1 ratio. Here we conducted a safety study in cynomolgus macaques (cm) modeling our previous animal studies with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and β-subunits. Three doses (3.2 x 1012 vg (n=3), 3.2 x 1011 vg (n=2), or 1.1 x 1011 vg (n=2)) were tested with controls infused with vehicle (n=1), or transgene empty AAVrh8 vector at the highest dose (n=2). Most monkeys receiving AAVrh8-cmHexα/β developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose-dependent with the highest dose cohort producing symptoms within a month of infusion. One monkey in the lowest dose cohort was behaviorally asymptomatic but had MRI abnormalities in thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/β showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome and this study demonstrates the variations in safety profiles of AAVrh8-Hex α/β intracranial injection among different species despite encoding for self-proteins.

Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

The tick-borne flavivirus, Powassan virus (POWV) causes life-threatening encephalitis in humans in North America and Europe. POWV is transmitted by ixodid tick vectors that feed on small to medium-sized mammals, such as Peromyscus leucopus mice, which may serve as either reservoir, bridge or amplification hosts. Intraperitoneal and intracranial inoculation of 4-week old Peromyscus leucopus mice with 103 PFU of POWV did not result in overt clinical signs of disease. However, following intracranial inoculation, infected mice seroconverted to POWV and histopathological examinations revealed that the mice uniformly developed mild lymphocytic perivascular cuffing and microgliosis in the brain and spinal cord from 5 to 15 days post infection (dpi), suggesting an early inflammatory response. In contrast, intracranial inoculation of 4-week old C57BL/6 and BALB/c mice was lethal by 5 dpi. Intraperitoneal inoculation was lethal in BALB/c mice, but 40% (2/5) of C57BL/6 mice survived. We concluded that Peromyscus leucopus mice infected i.c. with a lethal dose of POWV support a limited infection, restricted to the central nervous system and mount an antibody response to the virus. However, they fail to develop clinical signs of disease and are able to control the infection. These results suggest the involvement of restriction factors, and the mechanism by which Peromyscus leucopus mice restrict POWV infection remains under study.

Neural networks that control reproduction must integrate social and hormonal signals, tune motivation, and coordinate social interactions. However, the neural circuit mechanisms for these processes remain unresolved. The medial preoptic area (mPOA), an essential node for social behaviors, comprises molecularly diverse neurons with widespread projections. Here we identify a steroid-responsive subset of neurotensin (Nts)-expressing mPOA neurons that interface with the ventral tegmental area (VTA) to form a socially engaged reward circuit. Using in vivo two-photon imaging in female mice, we show that mPOANts neurons preferentially encode attractive male cues compared to nonsocial appetitive stimuli. Ovarian hormone signals regulate both the physiological and cue-encoding properties of these cells. Furthermore, optogenetic stimulation of mPOANts-VTA circuitry promotes rewarding phenotypes, social approach and striatal dopamine release. Collectively, these data demonstrate that steroid-sensitive mPOA neurons encode ethologically relevant stimuli and co-opt midbrain reward circuits to promote prosocial behaviors critical for species survival.

In mice, implantation always occurs towards the antimesometrial side of the uterus, while the placenta develops at the mesometrial side. What determines this particular orientation of the implanting blastocyst remains unclear. Uterine glands are critical for implantation and pregnancy. In this study, we showed that uterine gland development and active Wnt signalling activity is limited to the antimesometrial side of the uterus. Dkk2, a known antagonist of Wnt signalling, is only present at the mesometrial side of the uterus. Imaging of whole uterus, thick uterine sections (100-1000μm), and individual glands revealed that uterine glands are simple tubes with branches that are directly connected to the luminal epithelium and are only present towards the antimesometrial side of the uterus. By developing a unique mouse model targeting the uterine epithelium, we demonstrated that Wnt/β-catenin signaling is essential for prepubertal gland formation and normal implantation, but dispensable for postpartum gland development and regeneration. Our results for the first time have provided a probable explanation for the antimesometrial bias for implantation.

Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds.

An approximately 3,000 finishing swine operation in the United States experienced an outbreak of an atypical neurologic disease in 11-weeks-old pigs with an overall morbidity of 20% and case fatality rate of 30%. The clinical onset and progression of signs in affected pigs varied but included inappetence, compromised ambulation, ataxia, incoordination, mental dullness, paresis, paralysis and decreased response to environmental stimuli. Tissues from affected pigs were submitted for diagnostic investigation. Histopathologic examination of the cerebrum, cerebellum and spinal cord revealed severe lymphoplasmacytic and necrotizing polioencephalomyelitis with multifocal areas of gliosis and neuron satellitosis, suggestive of a neurotropic viral infection. Bacterial pathogens were not isolated by culture of neurologic tissue from affected pigs. Samples tested by polymerase chain reaction (PCR) were negative for pseudorabies virus and atypical porcine pestivirus. Immunohistochemistry for porcine reproductive and respiratory syndrome virus, porcine circovirus and Listeria was negative. Porcine sapelovirus (PSV) was identified in spinal cord by a nested PCR used to detect porcine enterovirus, porcine teschovirus and PSV. Next-generation sequencing of brainstem and spinal cord samples identified PSV and the absence of other or novel pathogens. In addition, Sapelovirus A mRNA was detected in neurons and nerve roots of the spinal cord by in situ hybridization. The PSV is genetically novel with an overall 94% amino acid identity and 86% nucleotide identity to a recently reported sapelovirus from Korea. This is the first case report in the United States associating sapelovirus with severe polioencephalomyelitis in pigs.

Ischemic heart disease resulting from myocardial infarction (MI) is the most prevalent form of heart disease in the United States. Post-MI cardiac remodeling is a multifaceted process that includes activation of fibroblasts and a complex immune response. T-regulatory cells (Tregs), a subset of CD4+ T cells, have been shown to suppress the innate and adaptive immune response and limit deleterious remodeling following myocardial injury. However, the mechanisms by which injured myocardium recruits suppressive immune cells remain largely unknown. Here, we have shown a role for Hippo signaling in the epicardium in suppressing the post-infarct inflammatory response through recruitment of Tregs. Mice deficient in epicardial YAP and TAZ, two core Hippo pathway effectors, developed profound post-MI pericardial inflammation and myocardial fibrosis, resulting in cardiomyopathy and death. Mutant mice exhibited fewer suppressive Tregs in the injured myocardium and decreased expression of the gene encoding IFN-γ, a known Treg inducer. Furthermore, controlled local delivery of IFN-γ following MI rescued Treg infiltration into the injured myocardium of YAP/TAZ mutants and decreased fibrosis. Collectively, these results suggest that epicardial Hippo signaling plays a key role in adaptive immune regulation during the post-MI recovery phase.

Villous cytotrophoblasts are epithelial stem cells of the early human placenta, able to differentiate either into syncytiotrophoblasts in floating chorionic villi or extravillous trophoblasts (EVTs) at the anchoring villi. The signaling pathways regulating differentiation into these two lineages are incompletely understood. The bulk of placental growth and development in the first trimester occurs under low oxygen tension. One major mechanism by which oxygen regulates cellular function is through the hypoxia-inducible factor (HIF), a transcription factor complex stabilized under low oxygen tension to mediate cellular responses, including cell fate decisions. HIF is known to play a role in trophoblast differentiation in rodents; however, its role in human trophoblast differentiation is poorly understood. Using RNA profiling of sorted populations of primary first-trimester trophoblasts, we evaluated the first stage of EVT differentiation, the transition from epidermal growth factor receptor+ villous cytotrophoblasts into human leukocyte antigen-G+ proximal column EVT (pcEVT) and identified hypoxia as a major pcEVT-associated pathway. Using primary cytotrophoblasts, we determined that culture in low oxygen directs differentiation preferentially toward human leukocyte antigen-G+ pcEVT, and that an intact HIF complex is required for this process. Finally, using global RNA profiling, we identified integrin-linked kinase and associated cytoskeletal remodeling and adhesion to be among HIF-dependent pcEVT-associated signaling pathways. Taken together, we propose that oxygen regulates EVT differentiation through HIF-dependent modulation of various cell adhesion and morphology-related pathways.

The role of human papillomavirus (HPV) as an etiologic and transformational agent in inverted Schneiderian papilloma (ISP) is unclear. Indeed, reported detection rates of HPV in ISPs range from 0 to 100%. The true incidence has been confounded by a tendency to conflate high- and low-risk HPV types and by the inability to discern biologically relevant from irrelevant HPV infections. The recent development of RNA in situ hybridization for high-risk HPV E6/E7 mRNA now allows the direct visualization of transcriptionally active high-risk HPV in ISP, providing an opportunity to more definitively assess its role in the development and progression of ISPs. We performed p16 immunohistochemistry and high-risk HPV RNA in situ hybridization on 30 benign ISPs, 7 ISPs with dysplasia, 16 ISPs with carcinomatous transformation, and 7 non-keratinizing squamous cell carcinomas (SCCs) with inverted growth that were unassociated with ISP. Transcriptionally active HPV was not detected in any of the 52 ISPs including those that had undergone carcinomatous transformation, but it was detected in two of seven (29%) non-keratinizing SCCs that showed inverted growth. There was a strong correlation between high-risk HPV RNA in situ hybridization and p16 immunohistochemistry (97%; p < 0.01). These results indicate that transcriptionally active high-risk HPV does not play a common role in either the development of ISP or in its transformation into carcinoma.

It is well-known that bone remodeling starts with a resorption event and ends with bone formation. However, what happens in between and how resorption and formation are coupled remains mostly unknown. Remodeling is achieved by so-called basic multicellular units (BMUs), which are local teams of osteoclasts, osteoblasts, and reversal cells recently proven identical with osteoprogenitors. Their organization within a BMU cannot be appropriately analyzed in common histology. The originality of the present study is to capture the events ranging from initiation of resorption to onset of formation as a functional continuum. It was based on the position of specific cell markers in longitudinal sections of Haversian BMUs generating new canals through human long bones. It showed that initial resorption at the tip of the canal is followed by a period where newly recruited reversal/osteoprogenitor cells and osteoclasts alternate, thus revealing the existence of a mixed "reversal-resorption" phase. 3D reconstructions obtained from serial sections indicated that initial resorption is mainly involved in elongating the canal and the additional resorption events in widening it. Canal diameter measurements show that the latter contribute the most to overall resorption. Of note, the density of osteoprogenitors continuously grew along the "reversal/resorption" surface, reaching at least 39 cells/mm on initiation of bone formation. This value was independent of the length of the reversal/resorption surface. These observations strongly suggest that bone formation is initiated only above a threshold cell density, that the length of the reversal/resorption period depends on how fast osteoprogenitor recruitment reaches this threshold, and thus that the slower the rate of osteoprogenitor recruitment, the more bone is degraded. They lead to a model where the newly recognized reversal/resorption phase plays a central role in the mechanism linking osteoprogenitor recruitment and the resorption-formation switch.

Abstract