Abstract
Aims
The NF2 gene encodes the tumour suppressor Merlin, which is deleted in 100% of patients with the familial tumour predisposition syndrome neurofibromatosis type 2 but also in 70% of ...those who develop sporadic schwannomas. The Raf-TR mouse model uses a tamoxifen-inducible Raf-kinase/ oestrogen receptor fusion protein (Raf-TR) expressed in myelinating Schwann cells to mimic a nerve injury response in Schwann cell by activating Raf/MEK/ERK signalling in the absence of peripheral nerve injury.
We will assess whether Raf/MEK/ERK activation on an NF2 null background leads to tumourigenesis within the vestibular nerves and dorsal root ganglia (DRGs), two tumour sites identified in the Periostin-Cre mouse model in which schwannoma formation is spontaneous, with a view to generating an inducible NF2 null schwannoma mouse model.
Method
Mice with a Schwann cell specific loss of Merlin were crossed with mice carrying a tamoxifen-inducible Raf-TR gene to generate Raf-TR+/-; P0-Cre+/-; NF2fl/fl (Cre+) mice which were NF2 null and compared to Raf-TR+/-; P0-Cre-/-; NF2fl/fl (Cre-) littermate controls. Mice were injected with tamoxifen or vehicle for five consecutive days and their vestibular nerves and dorsal root ganglia (DRGs) were analysed at various timepoints . An EdU proliferation assay was used to quantify the proliferation in the vestibular ganglia, as well as the DRGs. Rates of proliferation were compared to Cre- age-matched littermate controls treated with tamoxifen or vehicle.
Results
In the Periostin-Cre NF2 null schwannoma model, tumours form spontaneously in the DRGs and vestibular ganglia. In our new model, we see a clear increase in proliferation at 21 d post-injection in the NF2 null (Cre+) tamoxifen-treated mice compared to control (Cre-) tamoxifen-treated controls in both DRGs and vestibular ganglia. Cre- tamoxifen-treated mice do not show increased proliferation compared to Cre- vehicle controls. Taken together, this shows that activation of the Raf/MEK/ERK pathway in Schwann cells only causes a sustained proliferation response on an NF2 null background in the DRGs and vestibular ganglia. We are assessing later timepoints to further characterise tumour development in these mice.
Conclusion
Combining the Raf-TR mouse model to create a demyelinating phenotype with an NF2 null background leads to vastly increased rates of proliferation at the sites of schwannoma tumourigenesis within the peripheral nervous system: the DRGs and the vestibular ganglia. The high proliferation in the vestibular ganglia in particular is similar to the development of vestibular schwannomas in patients with Neurofibromatosis type 2. The new mouse model used in this study shows potential to be very useful as an inducible schwannoma tumour model, in which we can study the early events of tumour formation.
Abstract
Aims
Previous work has shown that increased numbers of macrophages are associated with more rapid schwannoma tumour growth and we are interested in signals that control entry of macrophages ...and other immune cells into these tumours. Activation of the Raf-kinase domain and the Raf/MEK/ERK pathway within Schwann cells has been observed to induce an inflammatory response in peripheral nerves in the absence of injury. Activation of an inducible Raf-kinase transgene in Schwann cells allows modelling of acute demyelination of peripheral nerves without nerve injury. This Raf-oestrogen receptor fusion protein (Raf-TR) is activated by the oestrogen analogue Tamoxifen and so allows targeted, controlled activation of the Raf/MEK/ERK pathway within the Schwann cells.
Here, in order to understand drivers of tumour formation, we assess the effect of MAPK activation in Merlin-null Schwann cells upon immune cell infiltration within the PNS.
Method
RafTR-P0CRE-NF2fl/fl mice of 4-6 weeks age were injected daily (IP) with 2mg of 4-hydroxy-tamoxifen or vehicle (corn oil) control for 5 consecutive days. RafTR was activated on either a Merlin (NF2) wild-type (NF2 fl/fl, P0-CRE-) or NF2 null (NF2 fl/fl, P0-CRE+) background and effects on immune cell infiltration studied in each condition.
Immunofluorescence was performed in the dorsal root ganglia (DRGs) and sciatic nerves of mice to identify various immune cell infiltrates at various timepoints. These will include neutrophils, mast cells, T-Cells and macrophages using the cell markers Csf3r, C-kit, CD3 and IBA1 respectively.
Results
At 21 days post treatment, a significantly increased infiltration of macrophages within the sciatic nerve and dorsal root ganglia was observed in mice treated with Tamoxifen when compared to vehicle controls. Loss of NF2 led to a massive increase in the number of macrophages recruited to peripheral nerves in tamoxifen-treated mice compared to Cre- mice and Cre+ treated with vehicle alone. Further assessment of other immune cell infiltration including neutrophils, mast cells and T cells are ongoing.
Conclusion
Raf/MEK/ERK signalling, in the absence of tumour suppressor Merlin, significantly increases the infiltration of inflammatory cells such as macrophages into peripheral nerves even in the absence of injury. As this effect is enhanced in NF2 null mice, this suggests that Merlin plays an important role in inhibiting the inflammatory response in peripheral nerves. It also suggests that Merlin could be involved in maintaining the blood nerve barrier (BNB), as in its absence the greater influx of immune cells into the nerves and DRGs suggests a more complete loss of BNB function than just activation of the Raf/MEK/ERK cascade alone.
Abstract
Aims
Our lab is interested in signals that trigger schwannoma tumour formation and we have previously shown that peripheral nerve injury triggers tumour formation in nerves with Schwann ...cell-specific loss of the Merlin (NF2) tumour suppressor. The Ras/Raf/MAPK/ERK pathway activity in myelinating Schwann cells is involved in nerve regeneration, causing demyelination and recruitment of inflammatory cells in areas of nerve damage, as well as dedifferentiation of myelinating Schwann cells into a repair-competent state. We have used a mouse model expressing a tamoxifen-inducible Raf-Kinase estrogen receptor fusion protein (Raf-TR) in myelinating Schwann cells of the PNS in either a control wild-type Merlin or Merlin-null background. This allows us to determine the effects of an injury-like signal in Schwann cells and its role in generating schwannoma tumour development. We present here a detailed analysis of the proliferation of Schwann cells within the nerve and morphological changes in PNS structure following Raf-TR activation.
Method
The P0-promotor driving the Raf-TR transgene is active in myelinating Schwann cells but inactive in the non-myelinating population, allowing specific targeting of the myelinating Schwann cell population. In addition to the Raf-TR gene, the mice exhibit a separate P0-promotor controlled Cre floxed NF2 gene which undergoes Cre-mediated recombinase at embryonic day 13.5 causing NF2 knockout in all developing Schwann cells. Mice aged between 4-6 weeks received intraperitoneal injections of either 2mg Tamoxifen or oil vehicle for 5 consecutive days and were then studied at either 10 or 21 days post-first injection. The peripheral nervous system of the mice was studied with fluorescent immuno-histochemistry staining, semithin sections and transmission electron microscopy (TEM) on sciatic nerves and dorsal root ganglia (DRG).
Results
Activation of the Ras/Raf/MAPK/ERK pathway in NF2 null Schwann cells led to higher rates of proliferation within sciatic nerves at 10d post-tamoxifen injections. At both 10d and 21d Raf-TR+ NF2-null mice sciatic nerve fascicles were visibly larger with significantly more cell bodies present than controls, however at 21d the rate of proliferation had reduced. In the DRG, proliferation was higher in Raf-TR+ NF2-null mice compared to controls, with proliferation remaining high at 21 days. Quantitative imaging of peripheral nerve semi-thins analysed to date showed no significant difference in the number of myelin rings present in the fascicles between different genotypes. Additionally, dual immuno-histochemistry staining with Myelin Basic Protein and EdU, markers for myelin and proliferation respectively, appeared to show proliferation in the non-myelinating Schwann cell population. Results from staining with other cell markers will also be presented, as well as a detailed analysis of nerve structure using TEM.
Conclusion
While developmental myelination of Merlin-null Schwann cells appears largely normal, the reaction of Merlin-null Schwann cells in the nerve to an injury signal (activation of the Raf-TR) is remarkably different from those of control nerves. The high levels of proliferation in Merlin-null Schwann cells may be indicative of a higher tumorigenesis potential. While the proliferation of Merlin-null cells does reduce over time in the sciatic nerve, further experiments are now testing whether there may be ongoing tumour growth at other locations in the nervous system that are associated with NF2 tumours in human patients.
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