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Using molecular signals controlling embryonic development to direct the differentiation of corticospinal motor neurons
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P.P. Kale, V. Addepalli / Int. J. Devl Neuroscience 30 (2012) 640–671
The dopaminergic stabilizer, (−)-OSU6162, rescues striatal neurons with normal and expanded polyglutamine chains in huntingtin protein from exposure to free radicals and mitochondrial toxins C. Ruiz 1,2 , M.J. Casarejos 2,3 , M.A. Mena 2,3,∗ , J.G. de Yebenes 1,2 1
Department of Neurology, Hospital Ramón y Cajal, Spain CIBERNED, Instituto de Salud Carlos III, Spain 3 Department of Neurobiology, Hospital Ramón y Cajal, Madrid, Spain 2
Huntington’s disease (HD) is a neurodegenerative disease characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and caused by a polyglutamine expansion in the huntingtin protein. There is so far neither cure nor approved disease-slowing therapy for HD, though recent clinical studies have shown a beneficial long-term effect of pridopidine in patients with HD. The nature of this effect, purely symptomatic or, in addition, neuroprotective, is difficult to elucidate in clinical trials. Pridopidine and (−)-OSU6162 are members of a new family of compounds referred to as dopaminergic stabilizers, which normalize abnormal dopamine neurotransmission. We investigated the effects of (−)-OSU6162 on huntingtin knocked-in striatal neurons in culture. Control neurons had normal full-length huntingtin with 7 glutamines while “mutant” neurons had large expansions (Q = 111). We studied the dose-effect curves of (−)-OSU6162 on mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and Q111 cells. In addition, we investigated the effects of (−)-OSU6162 on Q7 and Q111 neurons challenged with different neurotoxins such as sodium glutamate, H2 O2 , rotenone and 3-nitropropionic acid (3NP). As we found prevention of toxicity of some of these neurotoxins, we investigated the putative neuroprotective mechanisms of action of (−)-OSU6162 measuring the effects of this dopaminergic stabilizer on the ratios of Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the effects of (−)-OSU6162 on dopamine uptake and release. We found that (−)-OSU6162, 3-150 M, produces a dose dependent increase of mitochondrial activity and a reduction of cell death. (−)-OSU6162 does not change glutamate toxicity, but it partially prevents that of H2 O2 , rotenone and 3-nitropropionic acid. (−)-OSU6162 increases the levels Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells. (−)-OSU6162 increased 3 Hdopamine uptake and amphetamine-induced 3 H-dopamine release in E13 mouse mid brain neurons. Our studies demonstrate that (−)OSU6162 improves survival and mitochondrial function in striatal Q111 neurons and the resistance of these cells to several neurotoxins that damage striatal neurons, suggesting that (−)-OSU6162 and related compounds should be tested for neuroprotection in animal models and, eventually, in patients with HD. doi:10.1016/j.ijdevneu.2012.03.287
Using molecular signals controlling embryonic development to direct the differentiation of corticospinal motor neurons Youshan Melissa Lin 1,∗ , William Hendriks 2 , Paola Arlotta 1 1
Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, United States 2 Harvard Stem Cell Institute iPS Core Facility, Harvard University, United States The molecular signals that govern the development of distinct neuronal subtypes in the mammalian cerebral cortex are only beginning to be elucidated. Here, we focus on a specific subclass comprising corticospinal motor neurons (CSMN), a clinically rele-
vant neuronal population that degenerates in Amyotrophic Lateral Sclerosis (ALS), and are permanently damaged in spinal cord injury (SCI). With the identification of a series of neuronal subtype-specific genes that mark CSMN and other subcerebral projection neurons (SCPN) in vivo, it was notably discovered that the transcription factor Forebrain Embryonic Zinc Finger 2 (Fezf2) is necessary for the birth and early differentiation of all SCPN, including CSMN (i.e. Fezf2 −/− mice lack SCPN in their cortex), and is sufficient to “fate-switch” cortical progenitors fated to form upper layer callosal neurons to generate deep layer corticofugal projection neurons (CfuPN) that also includes CSMN. The central role played by Fezf2 in early SCPN differentiation led us to investigate whether cell-autonomous developmental signals that direct early SCPN development in the embryo could be used to selectively generate these neurons, from dorsal telencephalic progenitors derived from mouse induced pluripotent stem (miPS) cells, in vitro. We are currently testing whether overexpression of Fezf2 and other selected genes can progressively instruct the neuron type-specific differentiation of miPS cells into CfuPN, including CSMN. doi:10.1016/j.ijdevneu.2012.03.288
Nociceptive behaviour and lumbar spinal NADPH-d/NOS, p-ERK AND c-FOS expression after formalin pain induction in weaned rats Gila Behzadi Shahid Beheshti Med. Sci. University, Faculty of Medicine, Neuroscience Res. Ctr. And Physiology Department, Tehran, Iran Introduction: The sensitivity to formalin pain is an expression of neuronal plasticity in pain processing as the nervous system develops. Upon our previous study, the formalin pain responsiveness showed the complete appearance of phase II in 21 day-old weaned rats. In the present study we therefore were investigating the expression of Nitric Oxide Synthase by NADPH-d histochemistry and nNOS immunoreaction along with the p-ERK and c-FOS expression in lumbar spinal cord. Methods: Following right intraplantar injection of formalin solution (%10), coronal frozen section from L4–L5 were processed for each histochemical purposes. Results: 1–90 min after formalin injection we observed %25 and %30 increase in the number of NADPH-d and NOS containing neurons in lamina I-II of dorsal horn (p < 0.01). 2–5 min after formalin injection the number of p-ERK positive neurons were increased in lamina I-II significantly (p < 0.001). 3–120 min after formalin injection extensive number of c-FOS protein was expressed in lamina I–II neurons (p < 0.001). Discussion: Our results demonstrate a mature neuronal activation of lumbar spinal dorsal horn during phases I and II of formalin pain in weaned rats. doi:10.1016/j.ijdevneu.2012.03.289
P.P. Kale, V. Addepalli / Int. J. Devl Neuroscience 30 (2012) 640–671
The dopaminergic stabilizer, (−)-OSU6162, rescues striatal neurons with normal and expanded polyglutamine chains in huntingtin protein from exposure to free radicals and mitochondrial toxins C. Ruiz 1,2 , M.J. Casarejos 2,3 , M.A. Mena 2,3,∗ , J.G. de Yebenes 1,2 1
Department of Neurology, Hospital Ramón y Cajal, Spain CIBERNED, Instituto de Salud Carlos III, Spain 3 Department of Neurobiology, Hospital Ramón y Cajal, Madrid, Spain 2
Huntington’s disease (HD) is a neurodegenerative disease characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and caused by a polyglutamine expansion in the huntingtin protein. There is so far neither cure nor approved disease-slowing therapy for HD, though recent clinical studies have shown a beneficial long-term effect of pridopidine in patients with HD. The nature of this effect, purely symptomatic or, in addition, neuroprotective, is difficult to elucidate in clinical trials. Pridopidine and (−)-OSU6162 are members of a new family of compounds referred to as dopaminergic stabilizers, which normalize abnormal dopamine neurotransmission. We investigated the effects of (−)-OSU6162 on huntingtin knocked-in striatal neurons in culture. Control neurons had normal full-length huntingtin with 7 glutamines while “mutant” neurons had large expansions (Q = 111). We studied the dose-effect curves of (−)-OSU6162 on mitochondrial activity, LDH levels, necrosis and apoptosis in untreated Q7 and Q111 cells. In addition, we investigated the effects of (−)-OSU6162 on Q7 and Q111 neurons challenged with different neurotoxins such as sodium glutamate, H2 O2 , rotenone and 3-nitropropionic acid (3NP). As we found prevention of toxicity of some of these neurotoxins, we investigated the putative neuroprotective mechanisms of action of (−)-OSU6162 measuring the effects of this dopaminergic stabilizer on the ratios of Bcl2/Bax proteins and of p-ERK/ERK, the levels of chaperones and GSH, and the effects of (−)-OSU6162 on dopamine uptake and release. We found that (−)-OSU6162, 3-150 M, produces a dose dependent increase of mitochondrial activity and a reduction of cell death. (−)-OSU6162 does not change glutamate toxicity, but it partially prevents that of H2 O2 , rotenone and 3-nitropropionic acid. (−)-OSU6162 increases the levels Bcl2/Bax and decreases those of p-ERK/ERK and CHIP in Q111 cells. (−)-OSU6162 increased 3 Hdopamine uptake and amphetamine-induced 3 H-dopamine release in E13 mouse mid brain neurons. Our studies demonstrate that (−)OSU6162 improves survival and mitochondrial function in striatal Q111 neurons and the resistance of these cells to several neurotoxins that damage striatal neurons, suggesting that (−)-OSU6162 and related compounds should be tested for neuroprotection in animal models and, eventually, in patients with HD. doi:10.1016/j.ijdevneu.2012.03.287
Using molecular signals controlling embryonic development to direct the differentiation of corticospinal motor neurons Youshan Melissa Lin 1,∗ , William Hendriks 2 , Paola Arlotta 1 1
Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, United States 2 Harvard Stem Cell Institute iPS Core Facility, Harvard University, United States The molecular signals that govern the development of distinct neuronal subtypes in the mammalian cerebral cortex are only beginning to be elucidated. Here, we focus on a specific subclass comprising corticospinal motor neurons (CSMN), a clinically rele-
vant neuronal population that degenerates in Amyotrophic Lateral Sclerosis (ALS), and are permanently damaged in spinal cord injury (SCI). With the identification of a series of neuronal subtype-specific genes that mark CSMN and other subcerebral projection neurons (SCPN) in vivo, it was notably discovered that the transcription factor Forebrain Embryonic Zinc Finger 2 (Fezf2) is necessary for the birth and early differentiation of all SCPN, including CSMN (i.e. Fezf2 −/− mice lack SCPN in their cortex), and is sufficient to “fate-switch” cortical progenitors fated to form upper layer callosal neurons to generate deep layer corticofugal projection neurons (CfuPN) that also includes CSMN. The central role played by Fezf2 in early SCPN differentiation led us to investigate whether cell-autonomous developmental signals that direct early SCPN development in the embryo could be used to selectively generate these neurons, from dorsal telencephalic progenitors derived from mouse induced pluripotent stem (miPS) cells, in vitro. We are currently testing whether overexpression of Fezf2 and other selected genes can progressively instruct the neuron type-specific differentiation of miPS cells into CfuPN, including CSMN. doi:10.1016/j.ijdevneu.2012.03.288
Nociceptive behaviour and lumbar spinal NADPH-d/NOS, p-ERK AND c-FOS expression after formalin pain induction in weaned rats Gila Behzadi Shahid Beheshti Med. Sci. University, Faculty of Medicine, Neuroscience Res. Ctr. And Physiology Department, Tehran, Iran Introduction: The sensitivity to formalin pain is an expression of neuronal plasticity in pain processing as the nervous system develops. Upon our previous study, the formalin pain responsiveness showed the complete appearance of phase II in 21 day-old weaned rats. In the present study we therefore were investigating the expression of Nitric Oxide Synthase by NADPH-d histochemistry and nNOS immunoreaction along with the p-ERK and c-FOS expression in lumbar spinal cord. Methods: Following right intraplantar injection of formalin solution (%10), coronal frozen section from L4–L5 were processed for each histochemical purposes. Results: 1–90 min after formalin injection we observed %25 and %30 increase in the number of NADPH-d and NOS containing neurons in lamina I-II of dorsal horn (p < 0.01). 2–5 min after formalin injection the number of p-ERK positive neurons were increased in lamina I-II significantly (p < 0.001). 3–120 min after formalin injection extensive number of c-FOS protein was expressed in lamina I–II neurons (p < 0.001). Discussion: Our results demonstrate a mature neuronal activation of lumbar spinal dorsal horn during phases I and II of formalin pain in weaned rats. doi:10.1016/j.ijdevneu.2012.03.289