Adrenal Crisis as An Adverse Reaction to Zoledronic Acid in a Patient With Primary Adrenal Insufficiency: A Case Report and Literature Review.

Background/Objective: Adrenal crisis is a medical emergency and an acute complication of adrenal insufficiency (AI). It can be triggered by stressors such as infection, dehydration, trauma, or surgery. Case Report: We present a case of a 70-year-old woman with a history of Addison's disease, who presented in adrenal crisis within 24 hours after receiving her first infusion of zoledronic acid. No trigger was identified after extensive evaluation, making infusion with zoledronic acid the most likely cause of adrenal crisis. Discussion: Adverse reactions to medications can potentially trigger adrenal crisis. The present case report demonstrates that intravenous bisphosphonates can cause an acute phase reaction that may lead to adrenal crisis. Given the increased risk of osteoporosis in patients with AI there is an increased likelihood of prescription of intravenous bisphosphonates in this patient population. Conclusion: Patients with AI undergoing infusion with zoledronic acid may require an increased dose of glucocorticoid prior to infusion and may need to undergo monitoring post infusion for possible adrenal crisis.

Biphasic Roles of Hedgehog Signaling in the Production and Self-Renewal of Outer Radial Glia in the Ferret Cerebral Cortex.

The neocortex, the center for higher brain function, emerged in mammals and expanded in the course of evolution. The expansion of outer radial glia (oRGs) and intermediate progenitor cells (IPCs) plays key roles in the expansion and consequential folding of the neocortex. Therefore, understanding the mechanisms of oRG and IPC expansion is important for understanding neocortical development and evolution. By using mice and human cerebral organoids, we previously revealed that hedgehog (HH) signaling expands oRGs and IPCs. Nevertheless, it remained to be determined whether HH signaling expanded oRGs and IPCs in vivo in gyrencephalic species, in which oRGs and IPCs are naturally expanded. Here, we show that HH signaling is necessary and sufficient to expand oRGs and IPCs in ferrets, a gyrencephalic species, through conserved cellular mechanisms. HH signaling increases oRG-producing division modes of ventricular radial glia (vRGs), oRG self-renewal, and IPC proliferation. Notably, HH signaling affects vRG division modes only in an early restricted phase before superficial-layer neuron production peaks. Beyond this restricted phase, HH signaling promotes oRG self-renewal. Thus, HH signaling expands oRGs and IPCs in two distinct but continuous phases during cortical development.

Dysregulation of lysophospholipid signaling by p53 in malignant cells and the tumor microenvironment.

The TP53 gene has been widely studied for its roles in cell cycle control, maintaining genome stability, activating repair mechanisms upon DNA damage, and initiating apoptosis should repair mechanisms fail. Thus, it is not surprising that mutations of p53 are the most common genetic alterations found in human cancer. Emerging evidence indicates that dysregulation of lipid metabolism by p53 can have a profound impact not only on cancer cells but also cells of the tumor microenvironment (TME). In particular, intermediates of the sphingolipid and lysophospholipid pathways regulate many cellular responses common to p53 such as cell survival, migration, DNA damage repair and apoptosis. The majority of these cellular events become dysregulated in cancer as well as cell senescence. In this review, we will provide an account on the seminal contributions of Prof. Lina Obeid, who deciphered the crosstalk between p53 and the sphingolipid pathway particularly in modulating DNA damage repair and apoptosis in non-transformed as well as transformed cells. We will also provide insights on the integrative role of p53 with the lysophosphatidic acid (LPA) signaling pathway in cancer progression and TME regulation.

Plasma Epinephrine Contributes to the Development of Experimental Hypoglycemia-Associated Autonomic Failure.

BACKGROUND: Recurrent hypoglycemia blunts counter-regulatory responses to subsequent hypoglycemic episodes, a syndrome known as hypoglycemia-associated autonomic failure (HAAF). Since adrenergic receptor blockade has been reported to prevent HAAF, we investigated whether the hypoglycemia-associated rise in plasma epinephrine contributes to pathophysiology and reported interindividual differences in susceptibility to HAAF. METHODS: To assess the role of hypoglycemia-associated epinephrine responses in the susceptibility to HAAF, 24 adult nondiabetic subjects underwent two 2-hour hyperinsulinemic hypoglycemic clamp studies (nadir 54 mg/dL; 0-2 hours and 4-6 hours) on Day 1, followed by a third identical clamp on Day 2. We challenged an additional 7 subjects with two 2-hour infusions of epinephrine (0.03 µg/kg/min; 0-2 hours and 4-6 hours) vs saline on Day 1 followed by a 200-minute stepped hypoglycemic clamp (90, 80, 70, and 60 mg/dL) on Day 2. RESULTS: Thirteen out of 24 subjects developed HAAF, defined by ≥20% reduction in average epinephrine levels during the final 30 minutes of the third compared with the first hypoglycemic episode (P < 0.001). Average epinephrine levels during the final 30 minutes of the first hypoglycemic episode were 2.3 times higher in subjects who developed HAAF compared with those who did not (P = 0.006).Compared to saline, epinephrine infusion on Day 1 reduced the epinephrine responses by 27% at the 70 and 60 mg/dL glucose steps combined (P = 0.04), with a parallel reduction in hypoglycemic symptoms (P = 0.03) on Day 2. CONCLUSIONS: Increases in plasma epinephrine reproduce key features of HAAF in nondiabetic subjects. Marked interindividual variability in epinephrine responses to hypoglycemia may explain an individual's susceptibility to developing HAAF.

The LPA2 receptor agonist Radioprotectin-1 spares Lgr5-positive intestinal stem cells from radiation injury in murine enteroids.

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA2 GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA2, which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC50 that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by γ-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA2 either endogenously or after transfection. RP-1 reduced γ-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA2 GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after γ-irradiation. γ-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomatoflox transgenic mice, we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5+ intestinal stem cells. Taken together, our results suggest that the LPA2-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA2 GPCR in Lgr5+ stem cells.

mTORC1-Mediated Inhibition of 4EBP1 Is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma.

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice.

Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs) are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs) declines abruptly after postnatal maturation. We find that combining p27Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27Kip1, GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration.

ß-arrestin-2 is an essential regulator of pancreatic ß-cell function under physiological and pathophysiological conditions.

ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.

In Vivo Cochlear Hair Cell Generation and Survival by Coactivation of ß-Catenin and Atoh1.

The mammalian cochlea exhibit minimal spontaneous regeneration, and loss of sensory hair cells (HCs) results in permanent hearing loss. In nonmammalian vertebrates, spontaneous HC regeneration occurs through both proliferation and differentiation of surrounding supporting cells (SCs). HC regeneration in postnatal mammalian cochleae in vivo remains limited by the small HC number and subsequent death of regenerated HCs. Here, we describe in vivo generation of 10-fold more new HCs in the mouse cochlea than previously reported, most of which survive to adulthood. We achieved this by combining the expression of a constitutively active form of ß-catenin (a canonical Wnt activator) with ectopic expression of Atoh1 (a HC fate determination factor) in neonatal Lgr5+ cells (the presumed SC and HC progenitors of the postnatal mouse cochlea), and discovered synergistic increases in proliferation and differentiation. The new HCs were predominantly located near the endogenous inner HCs, expressed early HC differentiation markers, and were innervated despite incomplete alignment of presynaptic and postsynaptic markers. Surprisingly, genetic tracing revealed that only a subset of Lgr5+ cells that lie medial to the inner HCs respond to this combination, highlighting a previously unknown heterogeneity that exists among Lgr5+ cells. Together, our data indicate that ß-catenin and Atoh1 mediate synergistic effects on both proliferation and differentiation of a subset of neonatal cochlear Lgr5+ cells, thus overcoming major limitations of HC regeneration in postnatal mouse cochleae in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration. SIGNIFICANCE STATEMENT: Hearing loss in humans from aging, noise exposure, or ototoxic drugs (i.e., cisplatin or some antibiotics) is permanent and affects every segments of the population, worldwide. However, birds, frog, and fish have the ability to recover hearing, and recent studies have focused on understanding and applying what we have learned from them for restoring hearing in humans. However, studies have been hampered by low efficiency, limited cell numbers, and subsequent death of these newly generated auditory cells. Here, we describe a combinatorial approach, which results in the generation of auditory cells in greater numbers than previously reported, with most of them surviving to adult ages in vivo. These results provide a basis for combinatorial therapeutics for hearing restoration efforts.

Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.

Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells.

Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases.

Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea.

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of ß-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

Vagal neural crest cell migratory behavior: a transition between the cranial and trunk crest.

Migration and differentiation of cranial neural crest cells are largely controlled by environmental cues, whereas pathfinding at the trunk level is dictated by cell-autonomous molecular changes owing to early specification of the premigratory crest. Here, we investigated the migration and patterning of vagal neural crest cells. We show that (1) vagal neural crest cells exhibit some developmental bias, and (2) they take separate pathways to the heart and to the gut. Together these observations suggest that prior specification dictates initial pathway choice. However, when we challenged the vagal neural crest cells with different migratory environments, we observed that the behavior of the anterior vagal neural crest cells (somite-level 1-3) exhibit considerable migratory plasticity, whereas the posterior vagal neural crest cells (somite-level 5-7) are more restricted in their behavior. We conclude that the vagal neural crest is a transitional population that has evolved between the head and the trunk.

Regional differences in neural crest morphogenesis.

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.