Transcriptomic responses in mouse brain exposed to chronic excess of the neurotransmitter glutamate

Background: Increases during aging in extracellular levels of glutamate (Glu), the major excitatory neurotransmitter in the brain, may be linked to chronic neurodegenerative diseases. Little is known about the molecular responses of neurons to chronic, moderate increases in Glu levels. Genome-wide gene expression in brain hippocampus was examined in a unique transgenic (Tg) mouse model that exhibits moderate Glu hyperactivity throughout the lifespan, the neuronal Glutamate dehydrogenase (Glud1) mouse, and littermate 9 month-old wild type mice. Results: Integrated bioinformatic analyses on transcriptomic data were used to identify bio-functions, pathways and gene networks underlying neuronal responses to increased Glu synaptic release. Bio-functions and pathways up-regulated in Tg mice were those associated with oxidative stress, cell injury, inflammation, nervous system development, neuronal growth, and synaptic transmission. Increased gene expression in these functions and pathways indicated apparent compensatory responses offering protection against stress, promoting growth of neuronal processes (neurites) and re-establishment of synapses. The transcription of a key gene in the neurite growth network, the kinase Ptk2b, was significantly up-regulated in Tg mice as was the activated (phosphorylated) form of the protein. In addition to genes related to neurite growth and synaptic development, those associated with neuronal vesicle trafficking in the Huntington's disease signalling pathway, were also up-regulated. Conclusions: This is the first study attempting to define neuronal gene expression patterns in response to chronic, endogenous Glu hyperactivity at brain synapses. The patterns observed were characterized by a combination of responses to stress and stimulation of nerve growth, intracellular transport and recovery

Transcriptomic responses in mouse brain exposed to chronic excess of the neurotransmitter glutamate

<p>Abstract</p> <p>Background</p> <p>Increases during aging in extracellular levels of glutamate (Glu), the major excitatory neurotransmitter in the brain, may be linked to chronic neurodegenerative diseases. Little is known about the molecular responses of neurons to chronic, moderate increases in Glu levels. Genome-wide gene expression in brain hippocampus was examined in a unique transgenic (Tg) mouse model that exhibits moderate Glu hyperactivity throughout the lifespan, the neuronal <it>Glutamate dehydrogenase </it>(<it>Glud1</it>) mouse, and littermate 9 month-old wild type mice.</p> <p>Results</p> <p>Integrated bioinformatic analyses on transcriptomic data were used to identify bio-functions, pathways and gene networks underlying neuronal responses to increased Glu synaptic release. Bio-functions and pathways up-regulated in Tg mice were those associated with oxidative stress, cell injury, inflammation, nervous system development, neuronal growth, and synaptic transmission. Increased gene expression in these functions and pathways indicated apparent compensatory responses offering protection against stress, promoting growth of neuronal processes (neurites) and re-establishment of synapses. The transcription of a key gene in the neurite growth network, the kinase <it>Ptk2b</it>, was significantly up-regulated in Tg mice as was the activated (phosphorylated) form of the protein. In addition to genes related to neurite growth and synaptic development, those associated with neuronal vesicle trafficking in the Huntington's disease signalling pathway, were also up-regulated.</p> <p>Conclusions</p> <p>This is the first study attempting to define neuronal gene expression patterns in response to chronic, endogenous Glu hyperactivity at brain synapses. The patterns observed were characterized by a combination of responses to stress and stimulation of nerve growth, intracellular transport and recovery.</p

Ischemic Tolerance in an In Vivo Model of Glutamate “Preconditioning”

This is the peer reviewed version of the following article: Badawi, Y., Pal, R., Hui, D., Michaelis, E. K. and Shi, H. (2015), Ischemic tolerance in an in vivo model of glutamate preconditioning. Journal of Neuroscience Research, 93: 623–632. doi:10.1002/jnr.23517, which has been published in final form at http://doi.org/10.1002/jnr.23517. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. In this study, we focused on glutamate –mediated neuronal tolerance to ischemia-reperfusion. We employed an animal model of life-long excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo “glutamate preconditioning”. Nine- and 22-month old Tg and wild type (wt) mice were subjected to 90 min of middle cerebral artery occlusion followed by 24 hr reperfusion. The Tg mice suffered significantly reduced infarction and edema volume, compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and MAP2A expression to understand the mechanism of neuroprotection observed in the Tg Mice. We found that in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes while there were no significant differences in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising observation was that of a significant increase in MAP2A expression in neurons of the Tg hippocampus following ischemia-reperfusion, compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia-reperfusion

Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzenesulfonate (ABS) in tissues: a useful alternative to immunohistochemistry for fluorescence microscopy imaging of protein nitration

Protein tyrosine nitration is a common biomarker of biological aging and diverse pathologies associated with the excessive formation of reactive oxygen and nitrogen species. Recently, we suggested a novel fluorogenic derivatization procedure for the detection of 3-nitrotyrosine (3-NT) using benzylamine derivatives to convert specifically protein or peptide bound 3-NT to a highly fluorescent benzoxazole product. In the current study, we applied this procedure to fluorogenic derivatization of protein 3-NT in sections from adult rat cerebellum in order to: (i) test this method in imaging nitrated proteins in fixed brain tissue sections, and (ii) compare the chemical approach to immunohistochemical labeling with anti-3-NT antibodies. Immunofluorescence analysis of cerebellar sections using anti-3-NT antibodies showed differential levels of immunostaining in the molecular, Purkinje, and granule cell layers of the cerebellar cortex; in agreement with previous reports, the Purkinje cells were most highly labeled. Importantly, fluorogenic derivatization reactions of cerebellar proteins with 4-(aminomethyl)benzenesulfonic acid (ABS) and K3Fe(CN)6 at pH 9, following sodium dithionite (SDT) reduction of 3-NT to 3-aminotyrosine (3-AT), showed a very similar pattern of relative intensity of cell labeling and improved resolution when compared with antibody labeling. Our data demonstrate that ABS-derivatization may be either a useful alternative or a complimentary approach to immunolabeling in imaging protein nitration in cells and tissues, including under conditions of dual labeling with antibodies to cell proteins, thus allowing for cellular co-localization of nitrated proteins and any protein of interest

Genomic and biochemical approaches in the discovery of mechanisms for selective neuronal vulnerability to oxidative stress

Background: Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches. Results: In this report, using in vitro neuronal cultures, ex vivo organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between in vivo vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons. Conclusion: Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons

Neuronal Glud1 (Glutamate Dehydrogenase 1) Over-Expressing Mice: Increased Glutamate Formation and Synaptic Release, Loss of Synaptic Activity, and Adaptive Changes in Genomic Expression

Glutamate dehydrogenase 1 (GLUD1) is a mitochondrial enzyme expressed in all tissues, including brain. Although this enzyme is expressed in glutamatergic pathways, its function as a regulator of glutamate neurotransmitter levels is still not well defined. In order to gain an understanding of the role of GLUD1 in the control of glutamate levels and synaptic release in mammalian brain, we generated transgenic (Tg) mice that over-express this enzyme in neurons of the central nervous system. The Tg mice have increased activity of GLUD, as well as elevated levels and increased synaptic and depolarization-induced release of glutamate. These mice suffer age-associated losses of dendritic spines, nerve terminals, and neurons. The neuronal losses and dendrite structural changes occur in select regions of the brain. At the transcriptional level in the hippocampus, cells respond by increasing the expression of genes related to neurite growth and synapse formation, indications of adaptive or compensatory responses to the effects of increases in the release and action of glutamate at synapses. Because these Tg mice live to a relatively old age they are a good model of the effects of a “hyperglutamatergic” state on the aging process in the nervous system. The mice are also useful in defining the molecular pathways affected by the over-activation of GLUD in glutamatergic neurons of the brain and spinal cord

Transgenic Expression of Glud1 (Glutamate Dehydrogenase 1) in Neurons: In Vivo Model of Enhanced Glutamate Release, Altered Synaptic Plasticity, and Selective Neuronal Vulnerability

This is the published version. Copyright 2009 Society for Neuroscience.The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes

FEM Simulation of Non-Progressive Growth from Asymmetric Loading and Vicious Cycle Theory: Scoliosis Study Proof of Concept

Scoliosis affects about 1-3% of the adolescent population, with 80% of cases being idiopathic. There is currently a lack of understanding regarding the biomechanics of scoliosis, current treatment methods can be further improved with a greater understanding of scoliosis growth patterns. The objective of this study is to develop a finite element model that can respond to loads in a similar fashion as current spine biomechanics models and apply it to scoliosis growth. Using CT images of a non-scoliotic individual, a finite element model of the L3-L4 vertebra was created. By applying asymmetric loading in accordance to the ‘vicious cycle’ theory and through the use of a growth modulation equation it is possible to determine the amount of growth each region of the vertebra will undergo; therefore predict scoliosis growth over a period of time. This study seeks to demonstrate how improved anatomy can expand researchers current knowledge of scoliosis

Genomic and biochemical approaches in the discovery of mechanisms for selective neuronal vulnerability to oxidative stress

<p>Abstract</p> <p>Background</p> <p>Oxidative stress (OS) is an important factor in brain aging and neurodegenerative diseases. Certain neurons in different brain regions exhibit selective vulnerability to OS. Currently little is known about the underlying mechanisms of this selective neuronal vulnerability. The purpose of this study was to identify endogenous factors that predispose vulnerable neurons to OS by employing genomic and biochemical approaches.</p> <p>Results</p> <p>In this report, using <it>in vitro </it>neuronal cultures, <it>ex vivo </it>organotypic brain slice cultures and acute brain slice preparations, we established that cerebellar granule (CbG) and hippocampal CA1 neurons were significantly more sensitive to OS (induced by paraquat) than cerebral cortical and hippocampal CA3 neurons. To probe for intrinsic differences between <it>in vivo </it>vulnerable (CA1 and CbG) and resistant (CA3 and cerebral cortex) neurons under basal conditions, these neurons were collected by laser capture microdissection from freshly excised brain sections (no OS treatment), and then subjected to oligonucleotide microarray analysis. GeneChip-based transcriptomic analyses revealed that vulnerable neurons had higher expression of genes related to stress and immune response, and lower expression of energy generation and signal transduction genes in comparison with resistant neurons. Subsequent targeted biochemical analyses confirmed the lower energy levels (in the form of ATP) in primary CbG neurons compared with cortical neurons.</p> <p>Conclusion</p> <p>Low energy reserves and high intrinsic stress levels are two underlying factors for neuronal selective vulnerability to OS. These mechanisms can be targeted in the future for the protection of vulnerable neurons.</p