Post Doctoral Fellowships
Noriyuki Kishi, M.D., Ph.D.
Children's Hospital, Harvard Medical School
"Molecular Controls Over Differentiation of Cortical Projection Neurons from Neural Precursors: Normal and MECP2 -/-"
Mentor: Jeffrey Macklis, M.D., D.HST
2-Year Award: $110,000
Research Sponsor: Festival of Food and Wine
Final Report (November 2004)
Rett syndrome is a disease of brain development that causes mental retardation and autistic behavior in girls (one in every 10,000 to 15,000 girls), as well as in a small group of boys. Recent research has revealed that a defect in a gene called the methyl-CpG-binding protein 2 (MECP2), which encodes a protein that suppresses expression of other genes (transcriptional repressor), causes Rett syndrome. MECP2 mutant mice display several symptoms that are similar to those of Rett patients. Although it is evident that the symptoms of these mutant mice are attributable to the lack of the MECP2 gene in the brain, we know little about how the MECP2 gene works in the brain, and why mutation of the MECP2 gene causes predominantly neurological symptoms, despite the fact that the MECP2 gene is expressed throughout the entire body.
My work has added invaluable insights in the role of MECP2 in nerve cell maturation in the brain. I investigated how mutation in MECP2 gene may cause the symptoms of Rett syndrome using MECP2 genetically modified mice which lack MECP2 gene. First, I investigated which cells express MECP2 in the brain, using mouse brains and cells grown in dishes in the laboratory. I found that MECP2 is expressed in nerve cells (neurons), not in glia. Furthermore, my studies show that MECP2 is expressed in more mature neurons rather than in immature moving neuronal precursors, indicating that MECP2 is involved in the maturation and maintenance of neurons.
Although my data strongly indicate that MECP2 is involved in the maturation and maintenance of neurons, MECP2 has also been detected at low levels during embryonic development, and experiments using frog eggs showed that MECP2 controls production of neurons. However, the idea that MECP2 controls cell fate decisions does not mesh with the symptoms of Rett syndrome, because no report shows that there is decreased number of neurons in the patients. To investigate whether MECP2 is involved in events of initial neural development in mammals, I developed a mouse neural progenitor/„stem cell‰ culture system that can investigate these events. My results, using cells from MECP2 mutant mice, indicate that MECP2 mutation does not affect initial neural development events in mice, suggesting that MECP2 plays a different role in mammals (including humans) than it does in frogs.
I next investigated the thickness of the high level brain area called the cerebral cortex (or neocortex) in MECP2 mutant mice. I measured the thickness of the neocortex, which is composed of six layers and is responsible for high level cognitive functions. There is significant reduction of thickness in the neocortex of MECP2 mutant brains. Why do MECP2 mutant brains appear to have a thinner cortex? Two possibilities are 1) loss of neurons; and 2) reduced size and /or complexity of neurons. When I measured the cell density in each layer of each genotype, the cell densities of layers II/III, IV, V, and VI in MECP2 mutant mice are significantly higher than those in wild-type mice, suggesting that the reduced thickness of cortex in MECP2 mutant mice is due to reduced size of neurons, rather than to loss of neurons. In agreement with this hypothesis, I performed direct cellular analysis showing that pyramidal neurons in layer II/III in MECP2 mutant mice are smaller and their dendrites are less complex than those in wild-type mice.
In addition, I am finalizing neuronal transplantation experiments to further investigate potential abnormalities of both cell size and amount/quality of branches they send (dendritic complexity) by neurons of MECP2-null mice. By transplanting MECP2 mutant neuroblasts genetically labeled with a special genetic green color (GFP fluorescence) into wild-type brains, I can address whether the cell size and dendritic complexity of MECP2 mutant neurons is comparable or abnormal compared with wild-type neurons, and, if abnormal, determine whether this is due to MECP2 in neurons themselves, or dependent on the environment. Although some of the results are not yet final, transplanted MECP2 mutant layer II/III pyramidal neurons are smaller and less complex even in the wild-type environment than those of transplanted wild-type neurons, indicating that wild-type environment does not rescue the phenotype of transplanted MECP2 mutant neurons. Thus, MECP2 in neurons themselves is the central reason for their abnormalities.
Taken together, my data indicate that MECP2 is involved in the maintenance and maturation of brain neurons, including their connections, and the stabilization of neurons with long axons, rather than the early development or movement of neurons as the brain is initially formed. I am now beginning experiments to investigate these issues directly at the molecular level.
Noriyuki Kishi, M.D., Ph.D.Children's Hospital, Harvard Medical School
"Molecular Controls Over Differentiation of Cortical Projection Neurons from Neural Precursors: Normal and MECP2 -/-"
Mentor: Jeffrey Macklis, M.D., D.HST
2-Year Award: $110,000
Research Sponsor: Festival of Food and Wine
Final Report (November 2004)
Rett syndrome is a disease of brain development that causes mental retardation and autistic behavior in girls (one in every 10,000 to 15,000 girls), as well as in a small group of boys. Recent research has revealed that a defect in a gene called the methyl-CpG-binding protein 2 (MECP2), which encodes a protein that suppresses expression of other genes (transcriptional repressor), causes Rett syndrome. MECP2 mutant mice display several symptoms that are similar to those of Rett patients. Although it is evident that the symptoms of these mutant mice are attributable to the lack of the MECP2 gene in the brain, we know little about how the MECP2 gene works in the brain, and why mutation of the MECP2 gene causes predominantly neurological symptoms, despite the fact that the MECP2 gene is expressed throughout the entire body.
My work has added invaluable insights in the role of MECP2 in nerve cell maturation in the brain. I investigated how mutation in MECP2 gene may cause the symptoms of Rett syndrome using MECP2 genetically modified mice which lack MECP2 gene. First, I investigated which cells express MECP2 in the brain, using mouse brains and cells grown in dishes in the laboratory. I found that MECP2 is expressed in nerve cells (neurons), not in glia. Furthermore, my studies show that MECP2 is expressed in more mature neurons rather than in immature moving neuronal precursors, indicating that MECP2 is involved in the maturation and maintenance of neurons.
Although my data strongly indicate that MECP2 is involved in the maturation and maintenance of neurons, MECP2 has also been detected at low levels during embryonic development, and experiments using frog eggs showed that MECP2 controls production of neurons. However, the idea that MECP2 controls cell fate decisions does not mesh with the symptoms of Rett syndrome, because no report shows that there is decreased number of neurons in the patients. To investigate whether MECP2 is involved in events of initial neural development in mammals, I developed a mouse neural progenitor/„stem cell‰ culture system that can investigate these events. My results, using cells from MECP2 mutant mice, indicate that MECP2 mutation does not affect initial neural development events in mice, suggesting that MECP2 plays a different role in mammals (including humans) than it does in frogs.
I next investigated the thickness of the high level brain area called the cerebral cortex (or neocortex) in MECP2 mutant mice. I measured the thickness of the neocortex, which is composed of six layers and is responsible for high level cognitive functions. There is significant reduction of thickness in the neocortex of MECP2 mutant brains. Why do MECP2 mutant brains appear to have a thinner cortex? Two possibilities are 1) loss of neurons; and 2) reduced size and /or complexity of neurons. When I measured the cell density in each layer of each genotype, the cell densities of layers II/III, IV, V, and VI in MECP2 mutant mice are significantly higher than those in wild-type mice, suggesting that the reduced thickness of cortex in MECP2 mutant mice is due to reduced size of neurons, rather than to loss of neurons. In agreement with this hypothesis, I performed direct cellular analysis showing that pyramidal neurons in layer II/III in MECP2 mutant mice are smaller and their dendrites are less complex than those in wild-type mice.
In addition, I am finalizing neuronal transplantation experiments to further investigate potential abnormalities of both cell size and amount/quality of branches they send (dendritic complexity) by neurons of MECP2-null mice. By transplanting MECP2 mutant neuroblasts genetically labeled with a special genetic green color (GFP fluorescence) into wild-type brains, I can address whether the cell size and dendritic complexity of MECP2 mutant neurons is comparable or abnormal compared with wild-type neurons, and, if abnormal, determine whether this is due to MECP2 in neurons themselves, or dependent on the environment. Although some of the results are not yet final, transplanted MECP2 mutant layer II/III pyramidal neurons are smaller and less complex even in the wild-type environment than those of transplanted wild-type neurons, indicating that wild-type environment does not rescue the phenotype of transplanted MECP2 mutant neurons. Thus, MECP2 in neurons themselves is the central reason for their abnormalities.
Taken together, my data indicate that MECP2 is involved in the maintenance and maturation of brain neurons, including their connections, and the stabilization of neurons with long axons, rather than the early development or movement of neurons as the brain is initially formed. I am now beginning experiments to investigate these issues directly at the molecular level.