Final Report Summary - ENAC_IN_ANSCS (The role of epithelial sodium channels in adult neural stem cells.)
Current biomedicine aims to harvest the regenerative and restorative potential of native stem cells for new transplantation or cell-replacement therapies. Stem cells have the capacity to repetitively produce new differentiating daughter cells and in the same time self-renew to maintain their own pool. In the brain, the adult neural stem cells (aNSCs) reside in two discrete regions called “neurogenic niches”. One neurogenic niche lies in the hippocampus, a brain structure critical for memory and learning. The other niche is formed in the wall of the lateral ventricles, hollow cisterns in the brain filled with the cerebrospinal fluid (CSF). Regulation of aNSCs by genetic and paracrine means is well mapped (Summary Figure 1, Fig1), however, it is not known how and by what sensors aNSCs can detect changes in their surroundings to appropriately modulate proliferation and self-maintenance. This proposal aimed to test a hypothesis that a one specific type of transmembrane proteins that control flux of ions, the epithelial sodium channel (ENaC), may serve as molecular sensor for adult neural stem cell and that it may be regulating their physiology. The proposal aimed to address two major questions of the field (Fig2). First, can ion channels act as molecular sensors for aNSCs detecting environmental changes in the niche? Second, how can ion channels serve as detectors of these environmental changes?
To address the aforementioned questions, we sorted aNSCa and other cells types from the subventricular zone (SVZ), one of the neurogenic niches of adult brain. The gene microarrays of sorted cells revealed that among hundreds of ion channels and transmembrane receptors, only one as highly enriched in aNSCs over other cells types. It was the SCNN1A gene encoding the alpha subunit of ENaC. We confirmed the presence of ENaC in aNSCs and also in neuroblasts by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and by immunohistochemical staining.
When we pharmacologically blocked or genetically knocked-down ENaC in primary cell cultures from adult SVZ, we observed reduced cell proliferation and increased cell death in proliferative conditions. In differentiating conditions, blocking or knocking-down ENaC lead to reduced pro-neuronal differentiation. Taken together, this suggests that sodium currents via ENaC are critical for cell proliferation and differentiation (Fig3).
Because in vitro cell systems are prone to culturing artifacts, we tested the role of ENaC in vivo (Fig4). We generated a transgenic mouse line, in which ENaC can be genetically deleted (knocked-out, KO) specifically in aNSCs and their progeny. Because the genetically recombined aNSCs and their progeny express green fluorescent protein (GFP), they can be distinguished from other cells and their behavior followed at different time points. Similar to in vitro experiments, ablation of ENaC in aNSCs and their progeny reduced their proliferation in adult SVZ and to a certain degree also in the subgranular zone (SGZ), the other neurogenic zone of adult brain. This reduction in proliferation then resulted in fewer adult-born neurons in the olfactory bulb, the final destination point of SVZ-driven neurogenesis. These results support the conclusion that ENaC is critical for vital neurogenesis in adult brain and its modulation may be a potential target for pharmacological modulation of adult neurogenesis.
We wished to understand how is ENaC regulated in aNSCs (Fig5). Because the ionic channel is mechanosensitive in other native tissues such as kidneys, we subjected aNSCs in the brain to different shear stress generated by fluid flow. Increase in fluid flow (and thus in shear stress) increased proliferation in WT but not in ENaC KO animals suggesting ENaC is regulated by fluid shear stress to instruct aNSCs to proliferate. Our following calcium imaging experiments showed that elevated fluid shear stress increases intracellular calcium oscillations downstream of ENaC. In summary, we showed for the first time that adult neural stem cells in the brain can sense fluid shear stress in the lateral ventricles via ENaC and use the fluid flow as a environmental cue to modulate their proliferation.
Human adult neurogenesis has been implicated in learning and memory as well as in pathology of various diseases such as depression and Huntington’s disease. These neurological conditions represent substantial burden to the affected patients and to the health and social care systems. Thus, modulating of proliferation potential of aNSCs by ENaC-specific pharmacology may open a new field of targeted research to improve brain functions that are dependent on functional adult neurogenesis. However, ENaC blockers are commonly used as diuretics to treat certain types of hypertension. As a side effect, they may reduce proliferation of aNSCs and thus attenuate adult neurogenesis in humans. Such side-effects could be especially alarming since human SVZ adult neurogenesis in humans may involve striatum and thus cognitive functions.
To address the aforementioned questions, we sorted aNSCa and other cells types from the subventricular zone (SVZ), one of the neurogenic niches of adult brain. The gene microarrays of sorted cells revealed that among hundreds of ion channels and transmembrane receptors, only one as highly enriched in aNSCs over other cells types. It was the SCNN1A gene encoding the alpha subunit of ENaC. We confirmed the presence of ENaC in aNSCs and also in neuroblasts by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and by immunohistochemical staining.
When we pharmacologically blocked or genetically knocked-down ENaC in primary cell cultures from adult SVZ, we observed reduced cell proliferation and increased cell death in proliferative conditions. In differentiating conditions, blocking or knocking-down ENaC lead to reduced pro-neuronal differentiation. Taken together, this suggests that sodium currents via ENaC are critical for cell proliferation and differentiation (Fig3).
Because in vitro cell systems are prone to culturing artifacts, we tested the role of ENaC in vivo (Fig4). We generated a transgenic mouse line, in which ENaC can be genetically deleted (knocked-out, KO) specifically in aNSCs and their progeny. Because the genetically recombined aNSCs and their progeny express green fluorescent protein (GFP), they can be distinguished from other cells and their behavior followed at different time points. Similar to in vitro experiments, ablation of ENaC in aNSCs and their progeny reduced their proliferation in adult SVZ and to a certain degree also in the subgranular zone (SGZ), the other neurogenic zone of adult brain. This reduction in proliferation then resulted in fewer adult-born neurons in the olfactory bulb, the final destination point of SVZ-driven neurogenesis. These results support the conclusion that ENaC is critical for vital neurogenesis in adult brain and its modulation may be a potential target for pharmacological modulation of adult neurogenesis.
We wished to understand how is ENaC regulated in aNSCs (Fig5). Because the ionic channel is mechanosensitive in other native tissues such as kidneys, we subjected aNSCs in the brain to different shear stress generated by fluid flow. Increase in fluid flow (and thus in shear stress) increased proliferation in WT but not in ENaC KO animals suggesting ENaC is regulated by fluid shear stress to instruct aNSCs to proliferate. Our following calcium imaging experiments showed that elevated fluid shear stress increases intracellular calcium oscillations downstream of ENaC. In summary, we showed for the first time that adult neural stem cells in the brain can sense fluid shear stress in the lateral ventricles via ENaC and use the fluid flow as a environmental cue to modulate their proliferation.
Human adult neurogenesis has been implicated in learning and memory as well as in pathology of various diseases such as depression and Huntington’s disease. These neurological conditions represent substantial burden to the affected patients and to the health and social care systems. Thus, modulating of proliferation potential of aNSCs by ENaC-specific pharmacology may open a new field of targeted research to improve brain functions that are dependent on functional adult neurogenesis. However, ENaC blockers are commonly used as diuretics to treat certain types of hypertension. As a side effect, they may reduce proliferation of aNSCs and thus attenuate adult neurogenesis in humans. Such side-effects could be especially alarming since human SVZ adult neurogenesis in humans may involve striatum and thus cognitive functions.
