Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling.

Acinar cells are the principal secretory units of multiple exocrine organs. A single-cell, layered, lumenized acinus forms from a large cohort of epithelial progenitors that must initiate and coordinate three cellular programs of acinar specification, namely, lineage progression, secretion, and polarization. Despite this well-known outcome, the mechanism(s) that regulate these complex programs are unknown. Here, we demonstrate that neuronal-epithelial cross-talk drives acinar specification through neuregulin (NRG1)-ERBB3-mTORC2 signaling. Using single-cell and global RNA sequencing of developing murine salivary glands, we identified NRG1-ERBB3 to precisely overlap with acinar specification during gland development. Genetic deletion of Erbb3 prevented cell lineage progression and the establishment of lumenized, secretory acini. Conversely, NRG1 treatment of isolated epithelia was sufficient to recapitulate the development of secretory acini. Mechanistically, we found that NRG1-ERBB3 regulates each developmental program through an mTORC2 signaling pathway. Thus, we reveal that a neuronal-epithelial (NRG1/ERBB3/mTORC2) mechanism orchestrates the creation of functional acini.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1 -/- mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

Chromosomes missegregated into micronuclei contribute to chromosomal instability by missegregating at the next division.

Micronuclei (MNi) are extranuclear DNA-containing structures that form upon mitotic exit from unsegregated chromosome fragments or anaphase lagging (whole) chromosomes (LCs). MNi formed from whole chromosomes are of particular interest because LCs are observed in both cancer and non-cancer cells, and are recognized as a major source of chromosomal instability (CIN) in cancer cells. Here, we generated a PtK1 cell line expressing a photoactivatable H2B histone to study the behavior of whole chromosome-containing MNi at the mitosis following their formation. Importantly, MNi of PtK1 cells did not display the membrane rupture or transport defects reported for other cell types. Despite this, we found that most micronucleated cells displayed some kind of chromosome segregation defect and that the missegregating chromosome was the one derived from the MN. Moreover, condensation of the chromosome within the MN was frequently delayed and associated with failure to align at the metaphase plate. Finally, the defective condensation of the MN-derived chromosomes could also explain the frequent occurrence of cytokinesis failure in micronucleated cells. In summary, we find that chromosomes from MNi may trigger a CIN phenotype by missegregating at the mitosis following MN formation.

Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes.

Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1-/- adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.

Salivary glands regenerate after radiation injury through SOX2-mediated secretory cell replacement.

Salivary gland acinar cells are routinely destroyed during radiation treatment for head and neck cancer that results in a lifetime of hyposalivation and co-morbidities. A potential regenerative strategy for replacing injured tissue is the reactivation of endogenous stem cells by targeted therapeutics. However, the identity of these cells, whether they are capable of regenerating the tissue, and the mechanisms by which they are regulated are unknown. Using in vivo and ex vivo models, in combination with genetic lineage tracing and human tissue, we discover a SOX2+ stem cell population essential to acinar cell maintenance that is capable of replenishing acini after radiation. Furthermore, we show that acinar cell replacement is nerve dependent and that addition of a muscarinic mimetic is sufficient to drive regeneration. Moreover, we show that SOX2 is diminished in irradiated human salivary gland, along with parasympathetic nerves, suggesting that tissue degeneration is due to loss of progenitors and their regulators. Thus, we establish a new paradigm that salivary glands can regenerate after genotoxic shock and do so through a SOX2 nerve-dependent mechanism.

A novel antagonist of p75NTR reduces peripheral expansion and CNS trafficking of pro-inflammatory monocytes and spares function after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) results in long-term neurological deficits, which may be mediated in part by pro-inflammatory responses in both the injured brain and the circulation. Inflammation may be involved in the subsequent development of neurodegenerative diseases and post-injury seizures. The p75 neurotrophin receptor (p75NTR) has multiple biological functions, affecting cell survival, apoptotic cell death, axonal growth, and degeneration in pathological conditions. We recently found that EVT901, a novel piperazine derivative that inhibits p75NTR oligomerization, is neuroprotective, reduces microglial activation, and improves outcomes in two models of TBI in rats. Since TBI elicits both CNS and peripheral inflammation, we used a mouse model of TBI to examine whether EVT901 would affect peripheral immune responses and trafficking to the injured brain. METHODS: Cortical contusion injury (CCI)-TBI of the sensory/motor cortex was induced in C57Bl/6 wild-type mice and CCR2(+/RFP) heterozygote transgenic mice, followed by treatment with EVT901, a selective antagonist of p75NTR, or vehicle by i.p. injection at 4 h after injury and then daily for 7 days. Brain and blood were collected at 1 and 6 weeks after injury. Flow cytometry and histological analysis were used to determine peripheral immune responses and trafficking of peripheral immune cells into the lesion site at 1 and 6 weeks after TBI. A battery of behavioral tests administered over 6 weeks was used to evaluate neurological outcome, and stereological estimation of brain tissue volume at 6 weeks was used to assess tissue damage. Finally, multivariate principal components analysis (PCA) was used to evaluate the relationships between inflammatory events, EVT901 treatment, and neurological outcomes. RESULTS: EVT901 is neuroprotective in mouse CCI-TBI and dramatically reduced the early trafficking of CCR2+ and pro-inflammatory monocytes into the lesion site. EVT901 reduced the number of CD45(high)CD11b+ and CD45(high)F4/80+ cells in the injured brain at 6 weeks. TBI produced a significant increase in peripheral pro-inflammatory monocytes (Ly6C(int-high) pro-inflammatory monocytes), and this peripheral effect was also blocked by EVT901 treatment. Further, we found that blocking p75NTR with EVT901 reduces the expansion of pro-inflammatory monocytes, and their response to LPS in vitro, supporting the idea that there is a peripheral EVT901 effect that blunts inflammation. Further, 1 week of EVT901 blocks the expansion of pro-inflammatory monocytes in the circulation after TBI, reduces the number of multiple subsets of pro-inflammatory monocytes that enter the injury site at 1 and 6 weeks post-injury, and is neuroprotective, as it was in the rat. CONCLUSIONS: Together, these findings suggest that p75NTR signaling participates in the production of the peripheral pro-inflammatory response to CNS injury and implicates p75NTR as a part of the pro-inflammatory cascade. Thus, the neuroprotective effects of p75NTR antagonists might be due to a combination of central and peripheral effects, and p75NTR may play a role in the production of peripheral inflammation in addition to its many other biological roles. Thus, p75NTR may be a therapeutic target in human TBI.

Increased Gs Signaling in Osteoblasts Reduces Bone Marrow and Whole-Body Adiposity in Male Mice.

Bone is increasingly recognized as an endocrine organ that can regulate systemic hormones and metabolism through secreted factors. Although bone loss and increased adiposity appear to be linked clinically, whether conditions of increased bone formation can also change systemic metabolism remains unclear. In this study, we examined how increased osteogenesis affects metabolism by using an engineered G protein-coupled receptor, Rs1, to activate Gs signaling in osteoblastic cells in ColI(2.3)(+)/Rs1(+) transgenic mice. We previously showed that these mice have dramatically increased bone formation resembling fibrous dysplasia of the bone. We found that total body fat was significantly reduced starting at 3 weeks of age. Furthermore, ColI(2.3)(+)/Rs1(+) mice showed reduced O2 consumption and respiratory quotient measures without effects on food intake and energy expenditure. The mice had significantly decreased serum triacylglycerides, leptin, and adiponectin. Resting glucose and insulin levels were unchanged; however, glucose and insulin tolerance tests revealed increased sensitivity to insulin. The mice showed resistance to fat accumulation from a high-fat diet. Furthermore, ColI(2.3)(+)/Rs1(+) mouse bones had dramatically reduced mature adipocyte differentiation, increased Wingless/Int-1 (Wnt) signaling, and higher osteoblastic glucose utilization than controls. These findings suggest that osteoblasts can influence both local and peripheral adiposity in conditions of increased bone formation and suggest a role for osteoblasts in the regulation of whole-body adiposity and metabolic homeostasis.

Salivary gland development and disease.

Mammalian salivary glands synthesize and secrete saliva via a vast interconnected network of epithelial tubes attached to secretory end units. The extensive morphogenesis required to establish this organ is dependent on interactions between multiple cell types (epithelial, mesenchymal, endothelial, and neuronal) and the engagement of a wide range of signaling pathways. Here we describe critical regulators of salivary gland development and discuss how mutations in these impact human organogenesis. In particular, we explore the genetic contribution of growth factor pathways, nerve-derived factors and extracellular matrix molecules to salivary gland formation in mice and humans.

Chromosome mis-segregation and cytokinesis failure in trisomic human cells.

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.