Radiation Impairs Perineural Invasion by Modulating the Nerve Microenvironment

Perineural invasion (PNI) by cancer cells is an ominous clinical event that is associated with increased local recurrence and poor prognosis. Although radiation therapy (RT) may be delivered along the course of an invaded nerve, the mechanisms through which radiation may potentially control PNI remain undefined. murine sciatic nerve model was used to study how RT to nerve or cancer affects nerve invasion by cancer.Cancer cell invasion of the DRG was partially dependent on DRG secretion of glial-derived neurotrophic factor (GDNF). A single 4 Gy dose of radiation to the DRG alone, cultured with non-radiated cancer cells, significantly inhibited PNI and was associated with decreased GDNF secretion but intact DRG viability. Radiation of cancer cells alone, co-cultured with non-radiated nerves, inhibited PNI through predominantly compromised cancer cell viability. In a murine model of PNI, a single 8 Gy dose of radiation to the sciatic nerve prior to implantation of non-radiated cancer cells resulted in decreased GDNF expression, decreased PNI by imaging and histology, and preservation of sciatic nerve motor function.Radiation may impair PNI through not only direct effects on cancer cell viability, but also an independent interruption of paracrine mechanisms underlying PNI. RT modulation of the nerve microenvironment may decrease PNI, and hold significant therapeutic implications for RT dosing and field design for patients with cancers exhibiting PNI

A Class III Semaphorin (Sema3e) Inhibits Mouse Osteoblast Migration and Decreases Osteoclast Formation In Vitro

Originally identified as axonal guidance cues, semaphorins are expressed throughout many different tissues and regulate numerous non-neuronal processes. We demonstrate that most class III semaphorins are expressed in mouse osteoblasts and are differentially regulated by cell growth and differentiation: Sema3d expression is increased and Sema3e expression decreased during proliferation in culture, while expression of Sema3a is unaffected by cell density but increases in cultures of mineralizing osteoblasts. Expression of Sema3a, -3e, and -3d is also differentially regulated by osteogenic stimuli; inhibition of GSK3β decreased expression of Sema3a and -3e, while 1,25-(OH)2D3 increased expression of Sema3e. Parathyroid hormone had no effect on expression of Sema3a, -3b, or -3d. Osteoblasts, macrophages, and osteoclasts express the Sema3e receptor PlexinD1, suggesting an autocrine and paracrine role for Sema3e. No effects of recombinant Sema3e on osteoblast proliferation, differentiation, or mineralization were observed; but Sema3e did inhibit the migration of osteoblasts in a wound-healing assay. The formation of multinucleated, tartrate-resistant acid phosphatase–positive osteoclasts was decreased by 81% in cultures of mouse bone marrow macrophages incubated with 200 ng/mL Sema3e. Correspondingly, decreased expression of osteoclast markers (Itgb3, Acp5, Cd51, Nfatc1, CalcR, and Ctsk) was observed by qPCR in macrophage cultures differentiated in the presence of Sema3e. Our results demonstrate that class III semaphorins are expressed by osteoblasts and differentially regulated by differentiation, mineralization, and osteogenic stimuli. Sema3e is a novel inhibitor of osteoclast formation in vitro and may play a role in maintaining local bone homeostasis, potentially acting as a coupling factor between osteoclasts and osteoblasts

Alcohol Exposure Decreases CREB Binding Protein Expression and Histone Acetylation in the Developing Cerebellum

Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.We demonstrate that CREB binding protein (CBP) is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd) trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders

Robo2-Slit1 dependent cell-cell interactions mediate assembly of the trigeminal ganglion

Vertebrate cranial sensory ganglia, responsible for sensation of touch, taste and pain in the face and viscera, are composed of both ectodermal placode and neural crest cells. The cellular and molecular interactions allowing generation of complex ganglia remain unknown. Here, we show that proper formation of the trigeminal ganglion, the largest of the cranial ganglia, relies on reciprocal interactions between placode and neural crest cells in chick, as removal of either population resulted in severe defects. We demonstrate that ingressing placode cells express the Robo2 receptor and early migrating cranial neural crest cells express its cognate ligand Slit1. Perturbation of this receptor-ligand interaction by blocking Robo2 function or depleting either Robo2 or Slit1 using RNA interference disrupted proper ganglion formation. The resultant disorganization mimics the effects of neural crest ablation. Thus, our data reveal a novel and essential role for Robo2-Slit1 signaling in mediating neural crest–placode interactions during trigeminal gangliogenesis

Sema3E/Plexin-D1 Mediated Epithelial-to-Mesenchymal Transition in Ovarian Endometrioid Cancer

Cancer cells often employ developmental cues for advantageous growth and metastasis. Here, we report that an axon guidance molecule, Sema3E, is highly expressed in human high-grade ovarian endometrioid carcinoma, but not low-grade or other ovarian epithelial tumors, and facilitates tumor progression. Unlike its known angiogenic activity, Sema3E acted through Plexin-D1 receptors to augment cell migratory ability and concomitant epithelial-to-mesenchymal transition (EMT). Sema3E-induced EMT in ovarian endometrioid cancer cells was dependent on nuclear localization of Snail1 through activation of phosphatidylinositol-3-kinase and ERK/MAPK. RNAi-mediated knockdown of Sema3E, Plexin-D1 or Snail1 in Sema3E-expressing tumor cells resulted in compromised cell motility, concurrent reversion of EMT and diminished nuclear localization of Snail1. By contrast, forced retention of Snail1 within the nucleus of Sema3E-negative tumor cells induced EMT and enhanced cell motility. These results show that in addition to the angiogenic effects of Sema3E on tumor vascular endothelium, an EMT strategy could be exploited by Sema3E/Plexin-D1 signaling in tumor cells to promote cellular invasion/migration

The development of descending projections from the brainstem to the spinal cord in the fetal sheep

<p>Abstract</p> <p>Background</p> <p>Although the fetal sheep is a favoured model for studying the ontogeny of physiological control systems, there are no descriptions of the timing of arrival of the projections of supraspinal origin that regulate somatic and visceral function. In the early development of birds and mammals, spontaneous motor activity is generated within spinal circuits, but as development proceeds, a distinct change occurs in spontaneous motor patterns that is dependent on the presence of intact, descending inputs to the spinal cord. In the fetal sheep, this change occurs at approximately 65 days gestation (G65), so we therefore hypothesised that spinally-projecting axons from the neurons responsible for transforming fetal behaviour must arrive at the spinal cord level shortly before G65. Accordingly we aimed to identify the brainstem neurons that send projections to the spinal cord in the mature sheep fetus at G140 (term = G147) with retrograde tracing, and thus to establish whether any projections from the brainstem were absent from the spinal cord at G55, an age prior to the marked change in fetal motor activity has occurred.</p> <p>Results</p> <p>At G140, CTB labelled cells were found within and around nuclei in the reticular formation of the medulla and pons, within the vestibular nucleus, raphe complex, red nucleus, and the nucleus of the solitary tract. This pattern of labelling is similar to that previously reported in other species. The distribution of CTB labelled neurons in the G55 fetus was similar to that of the G140 fetus.</p> <p>Conclusion</p> <p>The brainstem nuclei that contain neurons which project axons to the spinal cord in the fetal sheep are the same as in other mammalian species. All projections present in the mature fetus at G140 have already arrived at the spinal cord by approximately one third of the way through gestation. The demonstration that the neurons responsible for transforming fetal behaviour in early ontogeny have already reached the spinal cord by G55, an age well before the change in motor behaviour occurs, suggests that the projections do not become fully functional until well after their arrival at the spinal cord.</p

Tumor interactions with soluble factors and the nervous system

In the genomic era of cancer research, the development of metastases has been attributed to mutations in the tumor that enable the cells to migrate. However, gene analyses revealed that primary tumors and metastases were in some cases genetically identical and the question was raised whether metastasis formation might be an inherent feature of certain tumor cells. In contradiction to this view, the last decade of cancer research has brought to light, that tumor cell migration, similar to leukocyte and fibroblast migration, is a highly regulated process. The nervous system plays an important role in this regulation, at least in two respects: firstly, neurotransmitters are known to regulate the migratory activity of tumor cells, and secondly, nerve fibers are used as routes for perineural invasion. We also summarize here the current knowledge on the innervation of tumors. Such a process might establish a neuro-neoplastic synapse, with the close interaction of tumor cells and nerve cells supporting metastasis formation

Axonal Varicosity Density as an Index of Local Neuronal Interactions

Diffuse transmission is an important non-synaptic communication mode in the cerebral neocortex, in which neurotransmitters released from en passant varicosities interact with surrounding cells. In a previous study we have shown that the cholinergic axonal segments which were in the microproximity with dopaminergic fibers possessed a greater density of en passant varicosities compared to more distant segments, suggesting an activity-dependent level of en passant varicosities in the axonal zone of interaction. To further evaluate this plastic relationship, the density of cholinergic varicosities was quantified on fiber segments within the microproximity of activated or non-activated pyramidal cells of the prefrontal cortex (mPFC). Repetitive 14 days patterned visual stimulation paired with an electrical stimulation of the cholinergic fibers projecting to the mPFC from the HDB was performed to induce persistent axonal plastic changes. The c-Fos early gene immunoreactivity was used as a neuronal activity marker of layer V pyramidal cells, labelled with anti-glutamate transporter EAAC1. Cholinergic fibers were labeled with anti-ChAT (choline acetyltransferase) immunostaining. The density of ChAT+ varicosities on and the length of fiber segments within the 3 µm microproximity of c-Fos positive/negative pyramidal cells were evaluated on confocal images. More than 50% of the pyramidal cells in the mPFC were c-Fos immunoreactive. Density of ChAT+ varicosities was significantly increased within 3 µm vicinity of activated pyramidal cells (0.50±0.01 per µm of ChAT+ fiber length) compared to non-activated cells in this group (0.34±0.001; p≤0.05) or control rats (0.32±0.02; p≤0.05). Different types of stimulation (visual, HDB or visual/HDB) induced similar increase of the density of ChAT+ varicosities within microproximity of activated pyramidal cells. This study demonstrated at the subcellular level an activity-dependent enrichment of ChAT+ varicosities in the axonal zone of interaction with other neuronal elements

Nos2 Inactivation Promotes the Development of Medulloblastoma in Ptch1+/− Mice by Deregulation of Gap43–Dependent Granule Cell Precursor Migration

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1+/− mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1+/− mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1+/+ Nos2−/− mice but not from Ptch1+/− Nos2−/− mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1+/+ Nos2−/− mice but increased in Ptch1+/− Nos2−/− mice relative to Ptch1+/− Nos2+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1+/− mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression