Impact of ionising radiation on adult neurogenesis: physiological and cellular effects of low and moderate doses of ionising radiation in neural differentiation distinguished by differentiation phase

Comprehensive follow-up of brain cancer patients and re-evaluation of medical data revealed a frightening correlation between low and moderate doses of ionising irradiation and induced cognitive dysfunctions. The particular radio sensitivity of adult neurogenesis within the adult brain is suggested as the major contributor in the underlying pathogenesis. Adult neurogenesis describes the differentiation of neural stem cells to mature neurons in the adult brain. Neural differentiation can be separated in three main phases, early progenitor phase, fate specification phase and cell maturation phase. The cellular base of neural differentiation provides a restricted stem cell pool, out of self-renewing neural stem cells (NSCs). To investigate the impact of low and moderate doses of ionising radiation (IR) on adult neurogenesis we intended to distinguish the differentiation process in its dynamic subpopulations, represented in the individual differentiation phases. Based on ES-derived NSCs we established and characterised a 2D-model system, reflecting the three differentiation phases, of adult neurogenesis. In the first part of this work we characterised the differentiation of the J1 NSC model system concerning specific marker expression, morphology and functional differentiation markers. The broad characterisation revealed that the J1 model system reflects each of the three differentiation phases, i.e. early progenitor, fate specification and cell maturation in adult neurogenesis on the intra- and intercellular as well as on the functional level in a synchronised, time dependent differentiation. We used the synchronous differentiation of the J1 model system to discriminate the individual differentiation phases by time. In the second part of this work we analysed the individual radio sensitivity of the distinct differentiation phases. Addressing issues concerning radiotherapy bystander and diagnostic doses, we used low to moderate x-ray doses between 0.25 and 2Gy. To estimate the distinct radio sensitivity of NSCs and the three differentiation phases, we measured the reduction in vitality of the separated subpopulations and estimated the individual LD50 of each differentiation phase. NSCs, as well as all three differentiation phases, show an individual radio sensitivity significantly different to the other subpopulations. The proliferative subpopulations, NSCs and early progenitors showed the highest radio sensitivity, whereas the final differentiation phase, cell maturation, showed the lowest. In further analyses we investigated the mechanisms responsible for the IR induced reductions in the individual subpopulations. Within the postmitotic differentiation phases, fate specification and cell maturation, radiation induced apoptosis is the underlying mechanism. In the proliferative subpopulations, the reduction in the number of cells is predominantly mediated by reduced proliferation in NSCs and induced apoptosis in early progenitors. To investigate the effect of IR on individual differentiation phases further, we irradiated the three defined differentiation phases and analysed the characteristic differentiation properties of the particular differentiation phase by functional, morphological and histochemical markers post IR. Early progenitors, the first subpopulation within the neural differentiation, are affected in their proliferation, but the migration of the surviving cells is not affected by low dose IR, neither is the entry into the postmitotic status. In the next step we analysed J1 cells irradiated in the fate specification phase. The fate determination of the surviving cell population showed a reduced percentage of future neurons post ionising radiation compared to unirradiated samples. The terminal differentiation phase revealed a broad modulation in the phase specific characteristic properties induced by low dose radiation, determined in altered neuronal architecture and a lower density of synaptic markers in neuroblasts as well as a reduced density of the voltage-gated potassium channels KV1.1. Regarding the functional level we found a long-lasting stagnation in the excitability of the in cell maturation phase irradiated cultures, reflected by a reduced firing rate, decreased coordination in the activity pattern and a loss in spike synchrony. In the last part of this work we investigated whether the properties of the self-renewing NSCs were also affected by IR. In previous work Dr. Bastian Roth determined that functional characteristics of J1 NSCs are modified post low dose IR, mediated via alterations in K+ channel currents. In continuative experiments we investigated if low dose IR leads to changes in the morphological and immunohistological characteristic within the J1 NSC population. The follow up of the irradiated NSCs revealed a highly significant increase of the differentiation marker doublecortin (DCX) within the self-renewing population. By using K+ channel blockers during radiation, we could inhibit the increase in DCX positive cells post low dose IR. In summary we found radiation induced modulations in the phase specific properties of each subpopulation, represented by NSCs and the three differentiation phases, in the third and fourth part of this work. In conclusion, depending on the differentiation phase, we could identify several specific physiological and cellular effects of low and moderate doses of IR during neural differentiation in a ES-derived neural stem cell line