Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are c... more Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.
The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly unders... more The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited b...
Amyloid-β precursor protein (APP) is well studied for its role in Alzheimer disease, although its... more Amyloid-β precursor protein (APP) is well studied for its role in Alzheimer disease, although its normal function remains uncertain. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem/progenitor cells (NSPCs). In this study we examined the role of APP in NSPC differentiation. To identify proteins that may mediate the effect of APP on NSPC differentiation, we used a gene array approach to find genes whose expression correlated with APP-induced neurogenesis. We found that the expression of neurogenin 2 (Ngn2), a basic helix-loop-helix transcription factor, was significantly down-regulated in NSPCs from APP knock-out mice (APPKO) and increased in APP transgenic (Tg2576) mice. Ngn2 overexpression in APPKO NSPCs promoted neuronal differentiation, whereas siRNA knockdown of Ngn2 expression in wild-type NSPCs decreased neuronal differentiation. The results demonstrate that APP-stimulated neuronal differentiation of NSPCs is mediated by Ngn2.
We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cr... more We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium, including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex, contribute multipotent, self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ, respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However, calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus, different SVZ stem cells have different embryonic origins, colonize different parts of the SVZ, and generate different neuronal progeny, suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future.
Although our understanding of adult neurogenesis has increased dramatically over the last decade,... more Although our understanding of adult neurogenesis has increased dramatically over the last decade, confusion still exists regarding both the identity of the stem cell responsible for neuron production and the mechanisms that regulate its activity. Here we show, using flow cytometry, that a small population of cells (0.3%) within the stem cell niche of the rat subventricular zone (SVZ) expresses the p75 neurotrophin receptor (p75(NTR)) and that these cells are responsible for neuron production in both newborn and adult animals. In the adult, the p75(NTR)-positive population contains all of the neurosphere-producing precursor cells, whereas in the newborn many of the precursor cells are p75(NTR) negative. However, at both ages, only the neurospheres derived from p75(NTR)-positive cells are neurogenic. We also show that neuron production from p75(NTR)-positive but not p75(NTR)-negative precursors is greatly enhanced after treatment with brain-derived neurotrophic factor (BDNF) or nerve growth factor. This effect appears to be mediated specifically by p75(NTR), because precursor cells from p75(NTR)-deficient mice show a 70% reduction in their neurogenic potential in vitro and fail to respond to BDNF treatment. Furthermore, adult p75(NTR)-deficient mice have significantly reduced numbers of PSA-NCAM (polysialylated neural cell adhesion molecule)-positive SVZ neuroblasts in vivo and a lower olfactory bulb weight. Thus, p75(NTR) defines a discrete population of highly proliferative SVZ precursor cells that are able to respond to neurotrophin activation by increasing neuroblast generation, making this pathway the most likely mechanism for the increased neurogenesis that accompanies raised BDNF levels in a variety of disease and behavioral situations.
Cytokines that signal through the LIFRbeta/gp130 receptor complex, including LIF and CNTF, promot... more Cytokines that signal through the LIFRbeta/gp130 receptor complex, including LIF and CNTF, promote the self-renewal of embryonic and adult neural precursor cells (NPCs). In non-CNS tissues, the protein suppressor of cytokine signaling-3 (SOCS3) negatively regulates signaling through gp130. Here, we analyze the role of SOCS3 in inhibiting LIF signaling in NPCs in vitro. SOCS3 is rapidly expressed by NPCs in response to LIF stimulation, with this expression largely dependent on recruitment of STAT proteins to the activated gp130 receptor. Proliferating NPC cultures can be generated from SOCS3 knockout (SOCS3KO/KO) embryos and display prolonged STAT3 phosphorylation and induction of the GFAP gene in response to LIF. In comparison with SOCS3 wild-type (SOCS3WT/WT) NPCs, SOCS3KO/KO cultures display enhanced self-renewal capacity. However, the clonal potential of SOCS3WT/WT but not SOCS3KO/KO NPCs is enhanced by exogenous LIF. Thus, SOCS3 acts as a negative regulator of LIF signaling in NPCs.
The number of cells generated by a proliferating stem or precursor cell can be influenced both by... more The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death-inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappaB (NFkappaB) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NFkappaB dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NFkappaB subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival.
Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are c... more Neuronal cytoskeletal alterations, in particular the loss and misalignment of microtubules, are considered a hallmark feature of the degeneration that occurs after traumatic brain injury (TBI). Therefore, microtubule-stabilizing drugs are attractive potential therapeutics for use following TBI. The best-known drug in this category is Paclitaxel, a widely used anti-cancer drug that has produced promising outcomes when employed in the treatment of various animal models of nervous system trauma. However, Paclitaxel is not ideal for the treatment of patients with TBI due to its limited blood-brain barrier (BBB) permeability. Herein we have characterized the effect of the brain penetrant microtubule-stabilizing agent Epothilone D (Epo D) on post-injury axonal sprouting in an in vitro model of CNS trauma. Epo D was found to modulate axonal sprout number in a dose dependent manner, increasing the number of axonal sprouts generated post-injury. Elevated sprouting was observed when analyzing the total population of injured neurons, as well as in selective analysis of Thy1-YFP-labeled excitatory neurons. However, we found no effect of Epo D on axonal sprout length or outgrowth speed. These findings indicate that Epo D specifically affects injury-induced axonal sprout generation, but not net growth. Our investigation demonstrates that primary cultures of cortical neurons are tolerant of Epo D exposure, and that Epo D significantly increases their regenerative response following structural injury. Therefore Epo D may be a potent therapeutic for enhancing regeneration following CNS injury. This article is part of a Special Issue entitled 'Traumatic Brain Injury'.
The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly unders... more The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited b...
Amyloid-β precursor protein (APP) is well studied for its role in Alzheimer disease, although its... more Amyloid-β precursor protein (APP) is well studied for its role in Alzheimer disease, although its normal function remains uncertain. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem/progenitor cells (NSPCs). In this study we examined the role of APP in NSPC differentiation. To identify proteins that may mediate the effect of APP on NSPC differentiation, we used a gene array approach to find genes whose expression correlated with APP-induced neurogenesis. We found that the expression of neurogenin 2 (Ngn2), a basic helix-loop-helix transcription factor, was significantly down-regulated in NSPCs from APP knock-out mice (APPKO) and increased in APP transgenic (Tg2576) mice. Ngn2 overexpression in APPKO NSPCs promoted neuronal differentiation, whereas siRNA knockdown of Ngn2 expression in wild-type NSPCs decreased neuronal differentiation. The results demonstrate that APP-stimulated neuronal differentiation of NSPCs is mediated by Ngn2.
We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cr... more We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium, including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex, contribute multipotent, self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ, respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However, calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus, different SVZ stem cells have different embryonic origins, colonize different parts of the SVZ, and generate different neuronal progeny, suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future.
Although our understanding of adult neurogenesis has increased dramatically over the last decade,... more Although our understanding of adult neurogenesis has increased dramatically over the last decade, confusion still exists regarding both the identity of the stem cell responsible for neuron production and the mechanisms that regulate its activity. Here we show, using flow cytometry, that a small population of cells (0.3%) within the stem cell niche of the rat subventricular zone (SVZ) expresses the p75 neurotrophin receptor (p75(NTR)) and that these cells are responsible for neuron production in both newborn and adult animals. In the adult, the p75(NTR)-positive population contains all of the neurosphere-producing precursor cells, whereas in the newborn many of the precursor cells are p75(NTR) negative. However, at both ages, only the neurospheres derived from p75(NTR)-positive cells are neurogenic. We also show that neuron production from p75(NTR)-positive but not p75(NTR)-negative precursors is greatly enhanced after treatment with brain-derived neurotrophic factor (BDNF) or nerve growth factor. This effect appears to be mediated specifically by p75(NTR), because precursor cells from p75(NTR)-deficient mice show a 70% reduction in their neurogenic potential in vitro and fail to respond to BDNF treatment. Furthermore, adult p75(NTR)-deficient mice have significantly reduced numbers of PSA-NCAM (polysialylated neural cell adhesion molecule)-positive SVZ neuroblasts in vivo and a lower olfactory bulb weight. Thus, p75(NTR) defines a discrete population of highly proliferative SVZ precursor cells that are able to respond to neurotrophin activation by increasing neuroblast generation, making this pathway the most likely mechanism for the increased neurogenesis that accompanies raised BDNF levels in a variety of disease and behavioral situations.
Cytokines that signal through the LIFRbeta/gp130 receptor complex, including LIF and CNTF, promot... more Cytokines that signal through the LIFRbeta/gp130 receptor complex, including LIF and CNTF, promote the self-renewal of embryonic and adult neural precursor cells (NPCs). In non-CNS tissues, the protein suppressor of cytokine signaling-3 (SOCS3) negatively regulates signaling through gp130. Here, we analyze the role of SOCS3 in inhibiting LIF signaling in NPCs in vitro. SOCS3 is rapidly expressed by NPCs in response to LIF stimulation, with this expression largely dependent on recruitment of STAT proteins to the activated gp130 receptor. Proliferating NPC cultures can be generated from SOCS3 knockout (SOCS3KO/KO) embryos and display prolonged STAT3 phosphorylation and induction of the GFAP gene in response to LIF. In comparison with SOCS3 wild-type (SOCS3WT/WT) NPCs, SOCS3KO/KO cultures display enhanced self-renewal capacity. However, the clonal potential of SOCS3WT/WT but not SOCS3KO/KO NPCs is enhanced by exogenous LIF. Thus, SOCS3 acts as a negative regulator of LIF signaling in NPCs.
The number of cells generated by a proliferating stem or precursor cell can be influenced both by... more The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death-inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappaB (NFkappaB) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NFkappaB dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NFkappaB subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival.
Uploads
Papers