PUB - Publikationen an der Universität Bielefeld

Protection of neurons against oxidative stress is crucial during neuronal development, maintenance and for the treatment of neurodegenerative diseases. However, little is known about the molecular mechanisms underlying sex-specific maturation and survival of neurons. In the present study, we demonstrated NF-kB-p65 mediated neuroprotection in human glutamatergic neurons differentiated from inferior turbinate stem cells (ITSCs) in a sex-dependent manner. For this we successfully differentiated ITSCs into MAP2+/NF200+/Synaptophysin+/vGlut2+-glutamatergic neurons in vitro and validated their functionality. TNF-α-dependent NF-kB-p65 activation was accompanied by significant neuroprotection against oxidative stress-induced neuronal death, which was unexpectedly higher in neurons from female donors. Accordingly, sex-specific neuroprotection of female neurons was followed by an increased expression of special NF-kB target genes SOD2 and IGF2. Among these, SOD2 is a well known gene protecting cells against oxidative stress resulting in longevity. While, IGF2 is known to promote synapse formation and spine maturation, and it has antioxidant and neuroprotective effects against oxidative damage. In addition, little is known about the role of NF-κB signaling in the regulation of neuronal differentiation particularly in human neural stem cells. In order to shed light on the understanding of NF-κB mechanisms during neuronal differentiation we efficiently differentiated ITSCs into glutamatergic and dopaminergic neurons as well, and we characterized their NF-κB subunit distribution during early neuronal differentiation, to establish the relevant subunits involved in their regulation. In this work, we demonstrated for the first time a major function of NF-κB during early human neuronal differentiation of ITSCs. We discovered that during glutamatergic differentiation, NF-κB pattern showed a nuclear activation of RelB/p52 heterodimers during an early phase, and a nuclear c-Rel activation peak during a later phase of differentiation. While the classical NF-κB subunits, RelA and p50 had no part during this differentiation. In contrast, during dopaminergic differentiation RelA had an important nuclear activity during an initial phase, whereas c-Rel showed a clear nuclear peak at a medial phase of differentiation, and RelA and RelB were both strongly increased at a final phase of dopaminergic differentiation. These results confirmed that NF-κB activity is strictly regulated during neuronal differentiation, and thus NF-κB-signalling might have a particular temporal pattern during the differentiation towards different cell fates. Furthermore, our findings revealed that pentoxifylline directly induced a shift from glutamatergic fate towards the oligodendrocyte fate, due to c-Rel nuclear inhibition, and also demonstrated that c-Rel has a key role in cell survival and neuronal fate. These results strongly suggested that NF-κB-c-Rel is essentially necessary for glutamatergic fate commitment during ITSCs differentiation. Moreover, we further analyzed differences in the ability of female and male-derived ITSCs to differentiate into dopaminergic neurons, and we determined sex-specific differences in the neuronal complexity. In addition, we chemically stimulated dopaminergic neurons using TNF-α, 6-hydroxydopamine (6OHDA) and their combination, to study their functionality. We found out that TNF-α-treatment induced a significant NF-κB-p65 nuclear activation, demonstrating a crucial role of NF-κB in dopaminergic function. Furthermore, stimulation using 6OHDA, exhibited a strong alteration in the neuronal morphology in both sexes, however there was a higher death tendency in male-derived neurons, compared to their female counterparts. Although the death rate differences between female- and male-derived neurons were not significantly different, these trend is in line with gender differences observed in Parkinson's disease, where more men are diagnosed with the disease than women. Also, our results introduce a neuroprotective effect induced by the activation of NF-κB-p65 mediated by TNF-α-pretreatment upon 6-OHDA stimulation of dopaminergic neurons derived from both sexes. Further analysis needs to be done to demonstrate this neuroprotective effect, and also the potential sex-specific differences that might be related to it.