Neuronal activity represses oncologic transformation of cerebellar external granule cells in medulloblastoma

Medulloblastoma (MB) is the most prevalent, metastatic pediatric brain tumor and has the highest mortality rate for childhood cancers. In Canada alone, the 25 – 50 children that are diagnosed with MB annually account for 15-25% of all pediatric brain tumours. This research focuses on the SHH MB subgroup which is prevalent in 30% of MB tumors. The cells of origin for SHH MB are cerebellar granule neuron precursors (cGNPs) in the external granule cell layer (EGL) on the surface of the cerebellum. During brain development, cGNPs migrate tangentially and actively proliferate in response to SHH, then migrate radially towards the internal granule cell layer (IGL) to differentiate. Our lab’s previous work demonstrated that a glutamate gradient, originating from the differentiated cells of the IGL may be diffusing towards undifferentiated cGNPs to initiate membrane depolarization, cell cycle exit, and differentiation. We hypothesized that neural activity impairs oncogenic transformation of cerebral granule neuron precursors and is tumor suppressive in SHH MB. We began by characterising the effects of neurotransmitters such as Glutamate and Gaba on cGNPs from P7 mice. The addition of Glutamate and GABA to cGNP cultures demonstrated an anti-proliferative effect despite the presence of a Shh agonist (SAG). Following this, glutamate production in P7 cerebellar cultures in vitro was measured to delineate the effect of glutamate on differential neural activity at different times of postnatal mouse cerebellum development. The results displayed a significant increase in glutamate production by P15 and P21 while simultaneously displaying glutamate’s presence in the post-natal brain as early as P5. Lastly, we measured spontaneous neural activity during development using RNA in-situ hybridization with c-fos and math1/atoh1 probes to understand whether that the in vivo glutamate gradient translates into increased neural activity. Early expression of the c-fos marker in undifferentiated cells demonstrated the presence of depolarising events in the EGL before synapses are formed in the IGL. Co-expression of math1 and c-fos in the int-EGL indicates the expression and manifestation of neural activity is an early event in differentiation in granule cells and the quantification of fluorescence using ImageJ compounded these results. Results from this thesis indicate that a glutamate gradient drives spontaneous neural activity in the cerebellum to counterbalance Shh induced proliferation and drive cell-cycle exit and differentiation

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