A replication-defective lentivirus vector that expresses enhanced green fluorescent protein (EGFP) under the control of a synthetic dopamine-beta-hydroxylase (DbetaH) promoter was used to define efferent projections of C1 catecholamine neurons in rat rostral ventrolateral medulla (RVLM). EGFP expression was restricted to C1 neurons and filled their somatodendritic compartments and efferent axons 7-28 days after vector injection. This included the descending projections to thoracic spinal cord and a network in brainstem, midbrain, and diencephalon. In caudal brainstem, restricted terminal fields were present in the dorsal motor vagal complex, A1, raphe pallidus and obscurus, and marginal layer of ventrolateral medulla. Innervation of raphe nuclei was most dense at the level of RVLM, but rostral levels of pallidus were devoid of innervation. A sparse commissural projection to contralateral RVLM was observed, and pericellular arbors were present in the dorsal reticular formation among the projection pathway of catecholamine axons. Rostral brainstem contained a dense innervation of locus coeruleus and the nucleus subcoeruleus. A restricted innervation of the ventrolateral column of the periaqueductal gray distinguished the midbrain. Forebrain labeling was restricted to the diencephalon, where distinctive terminal fields were observed in the paraventricular thalamic nucleus; the lateral hypothalamic area; and the paraventricular, dorsomedial, supraoptic, and median preoptic nuclei of hypothalamus. Projection fibers also coursed through the tuberal hypothalamus into the median eminence. Collectively, these data demonstrate that RVLM C1 neurons modulate the activity of other central cell groups known to participate in the regulation of cardiovascular and autonomic function.

Necrotizing enterocolitis (NEC) develops in response to elevated Toll-like receptor-4 (TLR4) signaling in the newborn intestinal epithelium, and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes, and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes(TLR4ΔIEC), and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared to full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7ΔIEC), the induction of autophagy was determined to be required for and not merely a consequence of NEC, as ATG7ΔIEC mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC, and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

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BACKGROUND MYCN amplification is a key molecular hallmark of high-risk neuroblastoma. Previously considered an “undruggable” target, MYCN transcription can be disrupted by inhibiting the bromodomain and extraterminal (BET) domain family of proteins that epigenetically regulates MYCN transcription. JQ1 is a potent small molecule BET inhibitor that has been shown to induce cell cycle arrest and to initiate apoptosis in neuroblastoma. Here, we sought to validate the anti-tumorigenic effects of JQ1 in neuroblastoma and to evaluate whether blocking N-myc expression with JQ1 promotes neural differentiation. METHODS We determined the effects of JQ1 treatment on human neuroblastoma in vitro cell growth in both monolayer and sphere-forming conditions. Subcutaneous neuroblastoma xenografts were used for in vivo study. Western blotting and immunohistochemistry were performed to evaluate for neural differentiation and stem cell markers. RESULTS JQ1 treatment blocked neuroblastoma cell growth in both monolayer and sphere-forming conditions; JQ1 also attenuated neuroblastoma xenograft growth. Neurofilament expression was enhanced with JQ1 treatment, indicating that JQ1 induces neuronal differentiation. Sphere forming conditions resulted in increased expression of stem cell markers; these were reversed with JQ1 treatment. CONCLUSIONS BET inhibition attenuates neuroblastoma progression and promotes neural differentiation, providing insight into clinical applications of BET inhibitors in the treatment of patients with neuroblastoma.