primary-culture-of-human-vestibular-schwannomas

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

University of Iowa Hospitals and Clinics

We investigated whether there are compensatory changes in the coronary microvasculature, cardiac lipid metabolism, and myocyte ultrastructure associated with ventricular enlargement in male rainbow trout. Epicardial tissue was sampled at different stages of sexual maturation, and we estimated arterial capillary density, intercapillary diffusion distance, and applied a diffusion model to predict PO(2) at different workloads. We also measured biochemical indices of lipid metabolism and estimated fractional volumes of mitochondria and myofibrils in myocytes. Immature fish with nonenlarged ventricles had the highest capillary length densities (1620+/-158 mm mm(-3)). Maturing trout with moderate ventricular hypertrophy had lower capillary length densities (1103+/-58 mm mm(-3)) and similar diffusion distances (13.9+/-0.7 microm) compared with immature fish (11.7+/-0.9 microm). The largest ventricles had intermediate capillary length densities (1457+/-288 mm mm(-3)) and diffusion distances (12.8+/-0.8 microm). Modelling predicted that enlarged ventricles would not become anoxic even at maximal workloads. Biochemical markers of fatty acid metabolism and aerobic capacity were unchanged with hypertrophy. Volume densities of mitochondria and myofibrils were also not influenced by cardiac growth. In summary, ventricle hypertrophy results in expansion of the coronary capillary bed and the maintenance of the epicardial capacities for fat and oxidative metabolism.

Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout.

After axotomy, Schwann cells (SCs), required for successful nerve regeneration, undergo a number of cellular changes including dedifferentiation, proliferation, expression of molecules that support axon growth, and apoptosis. This study investigated the role of p75, sortilin, and proneurotrophins in SC survival after facial nerve (FN) axotomy.

p75NTR and sortilin increase after facial nerve injury.

For vestibular schwannomas (VSs) that require treatment, options are limited to microsurgery or irradiation (IR). Development of alternative therapies that augment or replace microsurgery or IR would benefit patients not suitable for current therapies. This study explored the ability of ErbB2 inhibitors to modulate the effects of IR on VS cells.

Effects of ErbB2 signaling on the response of vestibular schwannoma cells to gamma-irradiation.

To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts.

The ErbB inhibitors trastuzumab and erlotinib inhibit growth of vestibular schwannoma xenografts in nude mice: a preliminary study.

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl-2 prevents SGN death but inhibits neurite growth. Here we assessed the consequences of Bcl-2 targeted to either the mitochondria (GFP-Bcl-2-Maob) or the endoplasmic reticulum (ER, GFP-Bcl-2-Cb5) on cultured SGN survival and neurite growth. Transfection of wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob increased SGN survival, with GFP-Bcl-2-Cb5 providing the most robust response. Paradoxically, expression of GFP-Bcl-2-Maob results in SGN death in the presence of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears to be specific for neurotrophin signaling, insofar as coexpression of constitutively active mitogen-activated kinase kinase (MEKDeltaEE) or phosphatidyl inositol-3 kinase (P110), but not other prosurvival stimuli (e.g., membrane depolarization), also results in the loss of SGNs expressing GFP-Bcl-2-Maob. MEKDeltaEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT-3 or MEKDeltaEE. However, wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5, and GFP-Bcl-2-Maob inhibit neurite growth even in the presence of neurotrophins, MEKDeltaEE, or P110. Historically, Bcl-2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl-2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl-2 targeted to the mitochondria results in SGN death in the presence of neurotrophins. (c) 2010 Wiley-Liss, Inc.

Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth.

Spiral ganglion Schwann cells (SGSCs) myelinate spiral ganglion neurons (SGNs) and represent a potential source of neurotrophic support for SGNs. Deafening due to loss of hair cells results in gradual degeneration and death of SGNs. Successful efforts to maintain or regenerate a functional auditory nerve may depend on a healthy population of SGSCs, yet the responses of SGSCs to neural injury remain largely unknown. Here we investigate the role of p75(NTR) in SGSC responses to gradual denervation. Following deafening, SGSCs in the osseous spiral lamina (OSL) and, subsequently, in Rosenthal's canal (RC) expressed elevated p75(NTR) compared to hearing controls. p75(NTR)-positive cells co-labeled with S100 and RIP antibodies (Schwann cell markers), but not with anti-neurofilament. The pattern of p75(NTR) expression mirrored the pattern of neural degeneration, beginning in the OSL of the cochlea base and later extending into the apex. SGSCs expressed sortilin, a p75(NTR) co-receptor for pro-neurotrophins. Both pro-nerve growth factor (pro-NGF) and pro-brain derived neurotrophic factor (proBDNF) induced apoptosis in cultured SGSCs. Deafened animals exhibited significantly higher levels of SGSC proliferation (as measured by BrdU uptake) compared to hearing animals while total Schwann cell density remained stable, suggesting a tight regulation of SGSC proliferation and cell death. SGSCs undergoing cell division lose p75(NTR) expression from the cell surface and demonstrate nuclear localization of the intracellular domain (ICD), raising the possibility that p75(NTR) cleavage and ICD nuclear localization regulate SGSC proliferation. These results suggest that p75(NTR) contributes to SGSC responses to deafening and neural degeneration.

p75(NTR) expression and nuclear localization of p75(NTR) intracellular domain in spiral ganglion Schwann cells following deafness correlate with cell proliferation.

Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase-signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.

Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides.

Regrowth of peripheral spiral ganglion neuron (SGN) fibers is a primary objective in efforts to improve cochlear implant outcomes and to potentially reinnervate regenerated hair cells. Cyclic adenosine monophosphate (cAMP) regulates neurite growth and guidance via activation of protein kinase A (PKA) and Exchange Protein directly Activated by Cylic AMP (Epac). Here we explored the effects of cAMP signaling on SGN neurite length in vitro. We find that the cAMP analog, cpt-cAMP, exerts a biphasic effect on neurite length; increasing length at lower concentrations and reducing length at higher concentrations. This biphasic response occurs in cultures plated on laminin, fibronectin, or tenascin C suggesting that it is not substrate dependent. cpt-cAMP also reduces SGN neurite branching. The Epac-specific agonist, 8-pCPT-2'-O-Me-cAMP, does not alter SGN neurite length. Constitutively active PKA isoforms strongly inhibit SGN neurite length similar to higher levels of cAMP. Chronic membrane depolarization activates PKA in SGNs and also inhibits SGN neurite length. However, inhibition of PKA fails to rescue neurite length in depolarized cultures implying that activation of PKA is not necessary for the inhibition of SGN neurite length by chronic depolarization. Expression of constitutively active phosphatidylinositol 3-kinase, but not c-Jun N-terminal kinase, isoforms partially rescues SGN neurite length in the presence of activated PKA. Taken together, these results suggest that activation of cAMP/PKA represents a potential strategy to enhance SGN fiber elongation following deafness; however such therapies will likely require careful titration so as to promote rather than inhibit nerve fiber regeneration.

Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons.

Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS.

Inhibition of c-Jun N-terminal kinase activity enhances vestibular schwannoma cell sensitivity to gamma irradiation.

Vestibular schwannomas (VSs) arise from Schwann cells (SCs) and result from the loss of function of merlin, the protein product of the NF2 tumor suppressor gene. In contrast to non-neoplastic SCs, VS cells survive long-term in the absence of axons. We find that p75(NTR) is overexpressed in VSs compared with normal nerves, both at the transcript and protein level, similar to the response of non-neoplastic SCs following axotomy. Despite elevated p75(NTR) expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75(NTR) . Furthermore, treatment with proNGF protects VS cells from apoptosis due to c-Jun N-terminal kinase (JNK) inhibition indicating that p75(NTR) promotes VS cell survival. Treatment of VS cells with proNGF activated NF-κB while inhibition of JNK with SP600125 or siRNA-mediated knockdown reduced NF-κB activity. Significantly, proNGF also activated NF-κB in cultures treated with JNK inhibitors. Thus, JNK activity appears to be required for basal levels of NF-κB activity but not for proNGF-induced NF-κB activity. To confirm that the increase in NF-κB activity contributes to the prosurvival effect of proNGF, we infected VS cultures with Ad.IκB.SerS32/36A virus, which inhibits NF-κB activation. Compared with control virus, Ad.IκB.SerS32/36A significantly increased apoptosis including in VS cells treated with proNGF. Thus, in contrast to non-neoplastic SCs, p75(NTR) signaling provides a prosurvival response in VS cells by activating NF-κB independent of JNK. Such differences may contribute to the ability of VS cells to survive long-term in the absence of axons. GLIA 2014.

J. Jason Clark

Department of Otolaryngology-Head and Neck

University of Iowa Hospitals and Clinics

Primary Culture of Human Vestibular Schwannomas