Final Report Summary - AGING STEM CELLS (Mechanisms of stem cell proliferation and senescence in the aged and damaged mouse brain)
Brain damage and neurodegeneration are two major causes of mortality and morbidity in the world. Additionally, the extremely restricted regenerative capacity of the adult brain has grea tly limited the development of new strategies that promote neuronal regeneration and recovery of brain function.
With the increase of life expectancy during the past decades, the health costs involving the treatment of neurodegenerative disorders has dramatically increased. There has been great scientific interest and effort on the development of stem cell-based therapies, but the biological mechanisms involved in the proliferation, survival and differentiation of neural stem cells are still largely unknown; many of these properties are also likely affected during ageing and brain damage, increasing the complexity of the study of stem cell-based therapies.
This work aimed to further understand the mechanisms involving the histone variant H2AX and the proliferation of stem cells in the ageing and damaged brain. It is known that neural stem cell proliferation decreases with age and cumulative telomere erosion activates DNA damage response proteins, including H2AX, which restrain the proliferative capacity of the stem cells. The role of H2AX in stem cell proliferation has been previously demonstrated by our laboratory, both in embryonic stem cells (ESC) (Andäng et al, Nature 2008) and in the subventricular zone (SVZ) of young adult mice (Fernando et al, PNAS 2011). In this work, we now observed that old animals lacking H2AX displayed a larger reduction in the number of stem cells in the SVZ than age-paired wild-type mice, suggesting that this histone may have a protective effect in the maintenance of stem cell number throughout life.
It is known that the stem cells of the SVZ ultimately give rise to new neurons that are integrated in the olfactory bulb circuitry, where they are involved in olfactory memory function. We then examined if the changes observed in the SVZ stem cells of the aged H2AX-deficient mice could result in alterations of the olfactory function. Our results showed that old H2AX-deficient mice seemed to have an impaired olfactory memory, as observed in an olfactory function behaviour test involving the recognition of new scents by the animals.
INK4a/Arf are critical regulators of cell cycle and induction of senescence. INK4a and Arf encode, respectively, the cyclin-dependent kinase inhibitor p16INK4a and p19Arf, which acts through inhibition the p53 inhibitory protein MDM2. Interestingly, both INK4a-/-/Arf-/- and H2AX-/- mice have similar alterations in stem cell proliferation. We then aimed to evaluate whether p16INK4a/p19Arf and H2AX interact in the control of stem cell proliferation and senescence, by crossing the INK4a-/-/Arf-/- mice (courtesy of Lene Uhrbom, Uppsala, Sweden) with our H2AX-/-. However, it was not feasible to obtain aged, triple knockout mice, as nearly all animals died before reaching one year of age, resulting in a not viable experiment in ethical terms. The quantification preliminary performed, however, revealed that both H2AX and INK4a/Arf can interact in the control of stem cell proliferation.
We then took advantage of two newly generated H2AX mutant mice to further study the importance of the activation of each residue in H2AX function: the H2AX-S139A mice, where the serine 139 is mutated to an alanine, and the H2AX-Y142F, where the tyrosine 142 is mutated to a phenylalanine. In both cases, the mutated H2AX proteins cannot be activated by phosphorylation. In the adult H2AX-S139A mutant mouse, we observed a decrease in the proliferation of stem cells in the SVZ, while no changes were detected in the H2AX-Y142F mouse. Both H2AX mutant animals were also used for the generation of primary embryonic fibroblast tissue culture, a simple system that allowed us to study the importance of each of the residues for cell proliferation. This allows us to investigate population growth and cell cycle progression of cells in a primary culture model that is technically more accessible. Our results showed that fibroblasts generated from H2AX-S139A embryos did not grow normally in culture, displaying what is likely a senescent state from the earliest culture time studied and maintained throughout subsequent passages. Additionally, these fibroblasts did not progress normally in the cell cycle, as shown by the decreased number of cells in S-phase and defective nucleoside incorporation rate. We hypothesize that removal of these cells from the embryo to a culture plate greatly reduced the proliferative ability of H2AX-S139A cells, possibly due to extracellular and/or tissue cues that were necessary to the growth of the fibroblasts. Importantly, these results suggest that H2AX-induced changes in cell proliferation may be highly dependent on tissue structure integrity. In contrast, the H2AX-Y142F fibroblasts had a normal growth curve and cell cycle profile, pointing to the specific requirement of serine 139 for cell cycle progression and proliferation.
In 2011, stroke was the cause of 6.2 million deaths in the world, the second most frequent cause of mortality. Additionally, 75% of those who survived a stroke episode will have a life-affecting disability. It is therefore of ultimate interest to study and develop new strategies to prevent stroke, as well as to reduce the morbidity associated with the incomplete recovery from a stroke. Therapies promoting the regeneration of the damaged tissue are clearly needed;despite much effort, there is no effective treatment for stroke patients.
Reports from our laboratory have previously demonstrated that GABAA receptors, via modulation of H2AX, affect stem cell proliferation in the mouse brain, which prompted us to investigate their potential in the regeneration of cells in the damaged brain. Namely, we investigated how a potential promoter of stem cell proliferation, the GABAA receptor antagonist bicuculline, could affect the neuronal outcome in a mouse stroke model. Bicuculline was administered one week prior to the stroke insult, and its impact on neuronal recovery was studied 2 and 7 weeks after the damage, allowing us to assess both the immediate and later influences of the early blockade of GABAA receptors upon neuronal recovery. We found that bicuculline treatment led to a higher number of immature neurons in the site of lesion 7 weeks post-stroke. These results suggest that early blockade of GABAA receptors can be beneficial in the recovery stage from a stroke episode.
Growth and repair outside the central nervous system are also heavily dependent on local sources of stem cells, and similarities in their properties and mechanisms can be observed among tissues. While the SVZ stem cells possess some characteristics of glial cells and express numerous glial markers, we recently published that peripheral nerve-associated glial cells are an important source of mesenchymal stem cells, which in turn are able to generate pulp cells and odontoblasts (Kaukua et al, Nature 2014). This highlights how stem cell differentiation properties and fate can in fact share common characteristics across different biological systems.
With the increase of life expectancy during the past decades, the health costs involving the treatment of neurodegenerative disorders has dramatically increased. There has been great scientific interest and effort on the development of stem cell-based therapies, but the biological mechanisms involved in the proliferation, survival and differentiation of neural stem cells are still largely unknown; many of these properties are also likely affected during ageing and brain damage, increasing the complexity of the study of stem cell-based therapies.
This work aimed to further understand the mechanisms involving the histone variant H2AX and the proliferation of stem cells in the ageing and damaged brain. It is known that neural stem cell proliferation decreases with age and cumulative telomere erosion activates DNA damage response proteins, including H2AX, which restrain the proliferative capacity of the stem cells. The role of H2AX in stem cell proliferation has been previously demonstrated by our laboratory, both in embryonic stem cells (ESC) (Andäng et al, Nature 2008) and in the subventricular zone (SVZ) of young adult mice (Fernando et al, PNAS 2011). In this work, we now observed that old animals lacking H2AX displayed a larger reduction in the number of stem cells in the SVZ than age-paired wild-type mice, suggesting that this histone may have a protective effect in the maintenance of stem cell number throughout life.
It is known that the stem cells of the SVZ ultimately give rise to new neurons that are integrated in the olfactory bulb circuitry, where they are involved in olfactory memory function. We then examined if the changes observed in the SVZ stem cells of the aged H2AX-deficient mice could result in alterations of the olfactory function. Our results showed that old H2AX-deficient mice seemed to have an impaired olfactory memory, as observed in an olfactory function behaviour test involving the recognition of new scents by the animals.
INK4a/Arf are critical regulators of cell cycle and induction of senescence. INK4a and Arf encode, respectively, the cyclin-dependent kinase inhibitor p16INK4a and p19Arf, which acts through inhibition the p53 inhibitory protein MDM2. Interestingly, both INK4a-/-/Arf-/- and H2AX-/- mice have similar alterations in stem cell proliferation. We then aimed to evaluate whether p16INK4a/p19Arf and H2AX interact in the control of stem cell proliferation and senescence, by crossing the INK4a-/-/Arf-/- mice (courtesy of Lene Uhrbom, Uppsala, Sweden) with our H2AX-/-. However, it was not feasible to obtain aged, triple knockout mice, as nearly all animals died before reaching one year of age, resulting in a not viable experiment in ethical terms. The quantification preliminary performed, however, revealed that both H2AX and INK4a/Arf can interact in the control of stem cell proliferation.
We then took advantage of two newly generated H2AX mutant mice to further study the importance of the activation of each residue in H2AX function: the H2AX-S139A mice, where the serine 139 is mutated to an alanine, and the H2AX-Y142F, where the tyrosine 142 is mutated to a phenylalanine. In both cases, the mutated H2AX proteins cannot be activated by phosphorylation. In the adult H2AX-S139A mutant mouse, we observed a decrease in the proliferation of stem cells in the SVZ, while no changes were detected in the H2AX-Y142F mouse. Both H2AX mutant animals were also used for the generation of primary embryonic fibroblast tissue culture, a simple system that allowed us to study the importance of each of the residues for cell proliferation. This allows us to investigate population growth and cell cycle progression of cells in a primary culture model that is technically more accessible. Our results showed that fibroblasts generated from H2AX-S139A embryos did not grow normally in culture, displaying what is likely a senescent state from the earliest culture time studied and maintained throughout subsequent passages. Additionally, these fibroblasts did not progress normally in the cell cycle, as shown by the decreased number of cells in S-phase and defective nucleoside incorporation rate. We hypothesize that removal of these cells from the embryo to a culture plate greatly reduced the proliferative ability of H2AX-S139A cells, possibly due to extracellular and/or tissue cues that were necessary to the growth of the fibroblasts. Importantly, these results suggest that H2AX-induced changes in cell proliferation may be highly dependent on tissue structure integrity. In contrast, the H2AX-Y142F fibroblasts had a normal growth curve and cell cycle profile, pointing to the specific requirement of serine 139 for cell cycle progression and proliferation.
In 2011, stroke was the cause of 6.2 million deaths in the world, the second most frequent cause of mortality. Additionally, 75% of those who survived a stroke episode will have a life-affecting disability. It is therefore of ultimate interest to study and develop new strategies to prevent stroke, as well as to reduce the morbidity associated with the incomplete recovery from a stroke. Therapies promoting the regeneration of the damaged tissue are clearly needed;despite much effort, there is no effective treatment for stroke patients.
Reports from our laboratory have previously demonstrated that GABAA receptors, via modulation of H2AX, affect stem cell proliferation in the mouse brain, which prompted us to investigate their potential in the regeneration of cells in the damaged brain. Namely, we investigated how a potential promoter of stem cell proliferation, the GABAA receptor antagonist bicuculline, could affect the neuronal outcome in a mouse stroke model. Bicuculline was administered one week prior to the stroke insult, and its impact on neuronal recovery was studied 2 and 7 weeks after the damage, allowing us to assess both the immediate and later influences of the early blockade of GABAA receptors upon neuronal recovery. We found that bicuculline treatment led to a higher number of immature neurons in the site of lesion 7 weeks post-stroke. These results suggest that early blockade of GABAA receptors can be beneficial in the recovery stage from a stroke episode.
Growth and repair outside the central nervous system are also heavily dependent on local sources of stem cells, and similarities in their properties and mechanisms can be observed among tissues. While the SVZ stem cells possess some characteristics of glial cells and express numerous glial markers, we recently published that peripheral nerve-associated glial cells are an important source of mesenchymal stem cells, which in turn are able to generate pulp cells and odontoblasts (Kaukua et al, Nature 2014). This highlights how stem cell differentiation properties and fate can in fact share common characteristics across different biological systems.