J. Jason Clark

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

Objective: To analyze the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts. Methods: Vestibular schwannoma xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor trastuzumab or saline for 2 weeks. Animals also received bromodeoxyuridine injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging during the treatment period. Cell death was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA. Results: Tumors can be distinguished with T2-weighted magnetic resonance imaging sequences. Trastuzumab significantly reduced the proliferation of VS cells compared with control (p < 0.01) as analyzed by bromodeoxyuridine uptake. Control tumors demonstrated slight growth during the 12-week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p < 0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA-positive VS cells (p < 0.01). Conclusion: In this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice, raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis Type 2.

p75NTR expression and nuclear localization of p75NTR intracellular domain in spiral ganglion Schwann cells following deafness correlate with cell proliferation

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 Rosenthals 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.

p75NTR and Sortilin Increase After Facial Nerve Injury

Objectives: 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 p75NTR, sortilin, and proneurotrophins in SC survival after facial nerve (FN) axotomy.

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

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 (MEKΔEE) 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. MEKΔEE and P110 promote SGN survival, whereas P110 promotes neurite growth to a greater extent than NT‐3 or MEKΔEE. However, wild‐type GFP‐Bcl‐2, GFP‐Bcl‐2‐Cb5, and GFP‐Bcl‐2‐Maob inhibit neurite growth even in the presence of neurotrophins, MEKΔEE, 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.

Fowler Award Presentation: Effects of ErbB2 Signaling on the Response of Vestibular Schwannoma Cells to γ‐Irradiation

Objective: 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.

Merlin status regulates p75NTR expression and apoptotic signaling in Schwann cells following nerve injury

After nerve injury, Schwann cells (SCs) dedifferentiate, proliferate, and support axon regrowth. If axons fail to regenerate, denervated SCs eventually undergo apoptosis due, in part, to increased expression of the low-affinity neurotrophin receptor, p75(NTR). Merlin is the protein product of the NF2 tumor suppressor gene implicated in SC tumorigenesis. Here we explore the contribution of merlin to SC responses to nerve injury. We find that merlin becomes phosphorylated (growth permissive) in SCs following acute axotomy and following gradual neural degeneration in a deafness model, temporally correlated with increased p75(NTR) expression. p75(NTR) levels are elevated in P0SchΔ39-121 transgenic mice that harbor an Nf2 mutation in SCs relative to wild-type mice before axotomy and remain elevated for a longer period of time following injury. Replacement of wild-type, but not phospho-mimetic (S518D), merlin isoforms suppresses p75(NTR) expression in primary human schwannoma cultures which otherwise lack functional merlin. Despite elevated levels of p75(NTR), SC apoptosis following axotomy is blunted in P0SchΔ39-121 mice relative to wild-type mice suggesting that loss of functional merlin contributes to SC resistance to apoptosis. Further, cultured SCs from mice with a tamoxifen-inducible knock-out of Nf2 confirm that SCs lacking functional merlin are less sensitive to p75(NTR)-mediated cell death. Taken together these results point to a model whereby loss of axonal contact following nerve injury results in merlin phosphorylation leading to increased p75(NTR) expression. Further, they demonstrate that merlin facilitates p75(NTR)-mediated apoptosis in SCs helping to explain how neoplastic SCs that lack functional merlin survive long-term in the absence of axonal contact.

p75NTR is highly expressed in vestibular schwannomas and promotes cell survival by activating nuclear transcription factor κB

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 p75NTR 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 p75NTR expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75NTR. Furthermore, treatment with proNGF protects VS cells from apoptosis due to c‐Jun N‐terminal kinase (JNK) inhibition indicating that p75NTR 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, p75NTR 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;62:1699–1712

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 either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Nf2 Mutation in Schwann Cells Delays Functional Neural Recovery Following Injury

Merlin is the protein product of the NF2 tumor suppressor gene. Germline NF2 mutation leads to neurofibromatosis type 2 (NF2), characterized by multiple intracranial and spinal schwannomas. Patients with NF2 also frequently develop peripheral neuropathies. While the role of merlin in SC neoplasia is well established, its role in SC homeostasis is less defined. Here we explore the role of merlin in SC responses to nerve injury and their ability to support axon regeneration. We performed sciatic nerve crush in wild-type (WT) and in P0SchΔ39-121 transgenic mice that express a dominant negative Nf2 isoform in SCs. Recovery of nerve function was assessed by measuring mean contact paw area on a pressure pad 7, 21, 60, and 90 days following nerve injury and by nerve conduction assays at 90 days following injury. After 90 days, the nerves were harvested and axon regeneration was quantified stereologically. Myelin ultrastructure was analyzed by electron microscopy. Functional studies showed delayed nerve regeneration in Nf2 mutant mice compared to the WT mice. Delayed neural recovery correlated with a reduced density of regenerated axons and increased endoneurial space in mutants compared to WT mice. Nevertheless, functional and nerve conduction measures ultimately recovered to similar levels in WT and Nf2 mutant mice, while there was a small (∼17%) reduction in the percent of regenerated axons in the Nf2 mutant mice. The data suggest that merlin function in SCs regulates neural ultrastructure and facilitates neural regeneration, in addition to its role in SC neoplasia.

p75NTRis highly expressed in vestibular schwannomas and promotes cell survival by activating nuclear transcription factor κB: p75NTRSignaling in Vestibular Schwannomas

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 p75NTR 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 p75NTR expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75NTR. Furthermore, treatment with proNGF protects VS cells from apoptosis due to c‐Jun N‐terminal kinase (JNK) inhibition indicating that p75NTR 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, p75NTR 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;62:1699–1712