Bruce K. Krueger - US grants

The principal goal of the proposed research is to isolate, reconstitute and characterize voltage-gated sodium channels from mammalian brain. These channels can be identified and assayed during purification by measuring the binding of radioactive ssxitoxin which specifically blocks the channels at nanomolar concentrations. Purification of saxitoxin binding sites from detergent-extracts of rat brain membranes will be carried out using standard procedures. The functional properties of toxin binding sites at each stage of purification will be determined by reconstitution of the binding sites in planar phospholipid bilayers for assay of voltage- and neurotoxin-dependent ion currents through the reconstituted channels. This method has been used to incorporate sodium channels from native neuronal membranes into planar bilayers; in both cases single channel current fluctuations and macroscopic (multichannel) currents were analyzed. This approach should minimize the possibility that important functional properties of the channels will be lost during biochemical manipulations of the binding sites. Once purified saxitoxin binding sites are obtained and reconstituted in planar bilayers, the effects of biochemical modifications including protein phosphorylation, on the polypeptide composition and on the physiological properties of the channels will be determined and compared. A second goal of this project is to use a combined electrophysiological and biochemical approach to characterize calcium-activated potassium channels from mammalian brain that have been reconsitituted in planar phospholipid bilayers. The possible roles of protein phosphorylation and of calmodulin in regulating the calcium-activated potassium channels will be evaluated. The long-range goal of this research program is to elucidate the molecular structures of ion channels in excitable membranes and to link specific structural components of the macromolecules with such functional properties of the channels as voltage-gating, ion permeation and calcium-activation.

The general goal of these studies is to gain an understanding of the conduction and gating of ionic currents through voltage sensitive sodium channels. To approach this goal, I will perform experiments on channels from mammalian brain incorporated into planar lipid bilayers as well as on voltage-clamped squid giant axons. Traditional bilayers formed between two aqueous chambers will be used when large membrane areas and easy access to solutions on both sides of the membrane are required. Micron-dimensioned bilayers formed on pipet tips, will enable the highest resolution of current records from single channels. The effects of incorporation into the artificial bilayers will be examined by comparing properties of incorporated channels with those observed by patch-clamping native membrane vesicles. Surprisingly, in initial studies of incorporated, batrachotoxin (BTX)-activated channels showed selectively near that of normal nerve. Hence, I plan a detailed evaluation of conduction in single BTX-treated channel, and subsequent studies in the absence of any activating alkaloid. I will take advantage of the ability offered by the bilayer system to separately manipulate fixed charges on the channel protein and the surrounding lipid. An attempt will be made to map the location of charges that affect either gating, ion permeation, or channel block by impermeant ions or toxins. Studies in squid axon have shown that addition of nonelectrolytes to the aqueous phase slows kinetics of gating and channel block, and reduces conductance. The mechanism of these changes will be investigated at the single channel level using the channels inserted into bilayers. Throughout this work, an underlying goal will be ultimately to develop the ability to measure single- and multi-channel ionic currents and gating currents in the same preparation. Experiments on squid axon will determine whether saxitoxin block of BTX-activated channels is voltage-dependent, as it is for brain channels in bilayers, as well as allow further comparison of native and incorporated channels.

Progressive neuronal degeneration underlies the cognitive dysfunction of Alzheimer's disease (AD). The neuritic plaque, consisting of deposits of Beta-amyloid (BetaA4) protein surrounded by reactive glia and degenerating neurons, is a hallmark of AD. Given the role of astrocytes (glial cells) in regulating the development and viability of neurons, the goal of this project is to test the hypothesis that abnormal glial-neuronal interactions influence neurodegeneration by altering neuronal Ca2+ homeostasis. One series of experiments will utilize the trisomy 16 (Ts16) mouse which contains an extra copy of chromosome 16, on which is located the gene coding for Beta-amyloid precursor protein (BetaAPP) and the mouse homolog of the putative familial AD locus. In order to test whether increased expression of one or more chromosome 16 genes affects neuronal properties, the effects of astrocytes and astrocyte-derived substances on the viability and structure of hippocampal neurons will be studied in vitro by co- culturing various combinations of Ts16 and euploid neurons and astrocytes, by examining effects of Ts16 and euploid astrocyte-conditioned medium on neuronal properties, and by adding various putative glial-derived growth factors and their neutralizing antibodies to the neuronal cultures. The role of intracellular Ca2+ in the premature death of Ts16 neurons in culture will be examined by computer-assisted imaging of ionized internal Ca2+ levels in Ts16 and euploid neurons using the fluorescent Ca2+ indicator, fura-2. A second series of experiments will study S100Beta, a Ca2+-binding protein widely distributed in brain in man, that is elevated in AD. S100Beta has been reported to have both neurotrophic and glial mitogenic activity. In order to test the hypothesis that excessive levels of S100Beta may overstimulate neuronal process formation, making the neuron more vulnerable to a loss of intracellular Ca2+ regulation, cultured hippocampal neurons from normal mice will be studied in the presence of S100Beta or anti-S100Beta antibodies. Neuronal viability and neurite configuration will be evaluated. The effects of S100Beta overexpression on astrocyte structure, function, and development will be investigated in cultured hippocampal astrocytes from transgenic S100Beta mice and in sections of transgenic S100Beta mouse brain. In addition, regulation of neuronal and astrocyte intracellular CA2+ in the presence of S100Beta will be studied by imaging of fura-2 fluorescence.

Progressive neuronal degeneration underlies the cognitive dysfunction of Alzheimer's disease (AD). The neuritic plaque, consisting of deposits of Beta-amyloid (BetaA4) protein surrounded by reactive glia and degenerating neurons, is a hallmark of AD. Given the role of astrocytes (glial cells) in regulating the development and viability of neurons, the goal of this project is to test the hypothesis that abnormal glial-neuronal interactions influence neurodegeneration by altering neuronal Ca2+ homeostasis. One series of experiments will utilize the trisomy 16 (Ts16) mouse which contains an extra copy of chromosome 16, on which is located the gene coding for Beta-amyloid precursor protein (BetaAPP) and the mouse homolog of the putative familial AD locus. In order to test whether increased expression of one or more chromosome 16 genes affects neuronal properties, the effects of astrocytes and astrocyte-derived substances on the viability and structure of hippocampal neurons will be studied in vitro by co- culturing various combinations of Ts16 and euploid neurons and astrocytes, by examining effects of Ts16 and euploid astrocyte-conditioned medium on neuronal properties, and by adding various putative glial-derived growth factors and their neutralizing antibodies to the neuronal cultures. The role of intracellular Ca2+ in the premature death of Ts16 neurons in culture will be examined by computer-assisted imaging of ionized internal Ca2+ levels in Ts16 and euploid neurons using the fluorescent Ca2+ indicator, fura-2. A second series of experiments will study S100Beta, a Ca2+-binding protein widely distributed in brain in man, that is elevated in AD. S100Beta has been reported to have both neurotrophic and glial mitogenic activity. In order to test the hypothesis that excessive levels of S100Beta may overstimulate neuronal process formation, making the neuron more vulnerable to a loss of intracellular Ca2+ regulation, cultured hippocampal neurons from normal mice will be studied in the presence of S100Beta or anti-S100Beta antibodies. Neuronal viability and neurite configuration will be evaluated. The effects of S100Beta overexpression on astrocyte structure, function, and development will be investigated in cultured hippocampal astrocytes from transgenic S100Beta mice and in sections of transgenic S100Beta mouse brain. In addition, regulation of neuronal and astrocyte intracellular CA2+ in the presence of S100Beta will be studied by imaging of fura-2 fluorescence.

This research program will investigate the relationship between neuron survival and intracellular Ca2+ homeostasis. We have found that both of these functions are defective in neurons from the trisomy 16 (Ts16) mouse. The Ts16 mouse has an extra copy of chromosome 16, the mouse homolog of human chromosome 21. Since patients with trisomy 21 (down syndrome) inevitable develop Alzeheimer's disease (AD), a neurodegenerative disorder characterized by neuronal death, understanding the mechanisms regulating Ts16 neuron survival may reveal abnormalities that contribute to AD. Using a novel in vitro assay for neuron survival, we have discovered that hippocampal neurons from the Ts16 mouse die 2-3 times faster than do normal (euploid) neurons. Our data demonstrate that survival of euploid neurons is promoted by micromolar concentrations of glutamate acting at kainate/AMPA receptors. Ts16 neurons lack this survival response to glutamate and this deficit can account for the accelerated death of Ts16 neurons. In contrast, both euploid and Ts16 neurons are rescued by peptide growth factors and killed by excitotoxic concentrations of added glutamate. Using computer-assisted fura-2 [Ca2+] imaging, we have also discovered that Ca2+ homeostasis is abnormal in both Ts16 neurons and astrocytes. We hypothesize that survival-promoting concentrations of glutamate maintain [Ca2+]cyt in an optimal range for euploid neuron survival and that this response is lacking in Ts16 neurons due to a genetically- determined defect in Ca2+ homeostasis. These hypotheses will be tested by correlating neuron survival with [Ca2+] in parallel experiments under conditions of varying degrees of survival. We propose experiments to determine the cellular mechanism underlying glutamate-promoted survival of normal neurons and the mechanistic basis for defective survival and Ca2+ homeostasis in Ts16 neurons. The Ts16 mouse is a naturally-occurring genetic defect that confers two discrete, but interested, deficits n normal cell physiology, viz., decreased neuronal survival and altered Ca2+ homeostasis. Both of these deficits may be masked in vivo by compensatory processes depending on cell type and cellular environment and, therefore, are most easily studied in vitro under well-controlled conditions. Deficits of this kind would be expedited to make cells vulnerable to toxic influences that accumulate with aging. Such vulnerability may play a role in the development of neurodegenerate disorders.

DESCRIPTION (adapted from applicant's abstract): The cause of mental retardation in Down syndrome (DS) is not understood but is thought to result, at least in part, from defective brain development during the embryonic period when neurons of the cerebral cortex are being generated. The trisomy 16 (Ts16) mouse shares a common genetic defect with DS and may be useful for studying the mechanisms underlying abnormal embryonic development of the cerebral cortex. Prenatal generation of postmitotic neurons in the Ts16 mouse cortex is delayed and subplate neurons are born concurrently with cortical plate neurons rather than preceding them as in the normal cortex. These abnormalities in the timing of Ts16 neurogenesis may lead to defective connectivity in the mature brain; similar defects during the prenatal development of the human brain may contribute to mental retardation in DS. Proliferation of neuroprogenitor cells (neuroblasts) and the decision of daughter cells to leave the cell cycle, which ultimately control the timing of neurogenesis, are regulated by neurotransmitters and growth factors such as glutamate and brain derived neurotrophic factor (BDNF). Ts16 neuroblasts fail to respond to glutamate and BDNF, raising the possibility that this signaling defect may underlie delayed neurogenesis in Ts16. The molecular basis for defects in the regulation of Ts16 neurogenesis will be studied in a) dissociated cell cultures of neuroblasts and b) organotypic slices from Ts16 and littermate euploid cortex. Both of these preparations enable not only the direct application of putative regulators of neurogenesis and of inhibitors of signaling pathways, but also direct measurement of proliferation, cell death and intracellular levels of Ca2+, a key modulator of proliferation, neuronal differentiation and migration. Experiments in organotypic slices will enable these processes to be analyzed in a structurally intact cortex and will allow the behavior of anatomically-distinct populations of neuroblasts and postmitotic neurons to be distinguished. The overall goals of this research project are to identify the signaling defects that lead to abnormal neurogenesis in the Ts16 mouse cerebral cortex and, at the same time, to determine the molecular signaling mechanisms underlying the control of neurogenesis in the normal brain.

DESCRIPTION (provided by applicant): Neurofibromatosis 1 (NF1) is an autosomal dominant disorder resulting from a spontaneous or inherited loss-of-function mutation in the gene encoding the regulatory protein, neurofibromin (NF). NF1 is characterized by tumors (neurofibromas) associated with the peripheral nervous system and by cognitive impairments including attention deficit hyperactivity disorder and learning and memory deficits. Although the genetic basis for NF1 has been established, the biological mechanisms by which loss of one copy of NF leads to the characteristic symptoms of the disease are not well understood. Studies with transgenic mice indicate that cognitive deficits are associated with the loss of GTPase activating protein (GAP) activity of NF. This research program will focus on the cellular and molecular mechanisms by which loss of NF GAP activity can lead to cognitive dysfunction and will test the hypothesis that the normal function of NF is to maintain low basal activity in the brain-derived neurotrophic factor (BDNF) signaling pathway by deactivating the G-protein, Ras. A prediction of this hypothesis is that partial loss of NF GAP activity in NF1 creates an abnormally high basal activity in the BDNF signaling pathway, leading to the dysregulation of BDNFmediated physiological functions underlying normal leaming and memory. This will be tested by examining BDNF signaling in genetically-modified neurons lacking NF. Although BDNF, via its cognate receptor, trkB, is known to activate multiple downstream pathways, not all of these should be affected by the loss of NF. Immunoprecipitation and proteomic analysis will be used to identify and characterize functional signaling complexes containing NF and trkB. If this hypothesis withstands the critical tests outlined in this proposal, the BDNF/trkB signaling pathway will emerge as a potential target for pharmacological or other therapies that could selectively treat the cognitive symptoms of NF1; the neurofibromas may be more effectively treated with a separate therapeutic strategy. In addition to testing this hypothesis, elucidation the components of the NF signaling complex will provide new insight into the normal function of NF and may provide additional clues to potential molecular targets for the treatment of NF1.

DESCRIPTION (provided by applicant): The neurotrophin, brain derived neurotrophic factor (BDNF), plays a critical role in brain development and function by regulating neuron survival and synaptic plasticity. Defects in BDNF signaling may lead to developmental, neurodegenerative and psychiatric disorders. Cellular responses to BDNF are mediated by the tyrosine receptor kinase, trkB, and their magnitude is determined by the relative levels of active, full-length trkB, and of truncated trkB isoforms, which act as dominant-negative inhibitors of BDNF signaling. Preliminary studies in cortical neurons revealed that expression of full-length trkB, but not that of truncated trkB, is stimulated by membrane depolarization via increased levels of intracellular Ca2+. Ca2+ was also found to exert opposing effects on the two promoters of the trkB gene (TRKB), suggesting that promoter usage regulates trkB isoform expression. These observations have led to the working hypothesis: TrkB isoform expression and consequently, neuronal responsiveness to BDNF, is regulated by Ca2+-dependent TRKB transcription. The mechanism by which Ca2+ differentially regulates the TRKB promoters will be studied using recombinant DNA technology to identify the Ca2+-dependent regulatory elements in TRKB and the transcription factors hat interact with them. The role of Ca2+-mediated promoter utilization in regulating the differential expression f full-length and truncated trkB will be studied by measuring the depolarization-induced expression f full-length and truncated trkB mRNA with promoter-specific 5'-untranslated sequences. In order to place Ca2+-dependent TRKB regulation in a more physiological context, we will investigate the ability of patterns of electrically evoked Ca2+ transients, which mimic in vivo neuronal activity, to differentially activate the TRKB promoters and differentially drive expression of full-length and truncated trkB isoforms. The long-term goal of this research is to elucidate the mechanisms by which Ca2+ regulates expression of trkB isoforms and, thus, modulates neuronal responsiveness to BDNF in the developing, adult and diseased brain.

DESCRIPTION (provided by applicant): The proper development of the nervous system during early pregnancy is particularly vulnerable to both environmental toxins and the effects of inherited genetic factors which can lead to errors in connectivity in the postnatal brain. Valproic acid (VPA) is an antiepileptic and mood stabilizing drug that, when administered during pregnancy, causes neurodevelopmental defects such as behavioral and cognitive dysfunction, including maladaptations observed in children with autism spectrum disorder and intellectual delay. The severity of effects appears to be dependent upon gestational time of maternal exposure. VPA is a histone deacetylase inhibitor, suggesting that it interferes with gene expression by an epigenetic mechanism. We have observed that administration of VPA to pregnant mice during early gestation increases the expression of brain-derived neurotrophic factor (BDNF), a neurotrophin that acts as a critical modulator of neurogenesis in the fetal brain. This has led to the working hypothesis for this proposal: epigenetic stimulation of BDNF expression by VPA during fetal brain development causes defective forebrain neurogenesis and behavioral deficits. This hypothesis will be tested by determining 1) the extent to which VPA-induced stimulation of BDNF expression is mediated by DNA methylation and/or covalent histone modification at specific BDNF gene promoters; 2) the extent to which the proportions of cortical pyramidal neurons and GABAergic interneurons are altered by embryonic exposure to VPA; and 3) the role of altered BDNF signaling, through the trkB receptor, in mediating the effects of VPA on embryonic forebrain neurogenesis and cognition. This will be accomplished utilizing a novel transgenic mouse with a mutant trkB receptor, engineered to be selectively and reversibly blocked by administration of an exogenous antagonist. The prediction for the latter experiments is that VPA will fail to induce neurogenetic defects and abnormal behavior when the BDNF signaling pathway is inhibited. The goal of this research is to determine the mechanism by which fetal exposure to a clinically used agent, VPA, induces neurodevelopmental defects. This would enable the identification of signaling pathways that can be targeted to avoid adverse neurodevelopmental effects in pregnant women who require VPA for control of epilepsy and bipolar disorder. In addition, the project seeks to establish a paradig that would enable systematic investigation of the mechanisms by which environmental agents affect brain development as well as how environmental and genetic factors might interact to cause autism and other neurodevelopmental disorders.

PROJECT SUMMARY The prevalence of neurodevelopmental disorders of behavior and cognition such as autism spectrum disorders (ASDs) is increased by prenatal exposure to pathogenic environmental stressors such as the anti-epileptic, mood-stabilizing drug, valproic acid (VPA) and organophosphate insecticides such as chlorpyrifos. The under- lying cellular and molecular mechanisms are not known. Many mental disorders are sexually dimorphic. Some (e.g., ASD and ADHD) are more common in males whereas others (e.g., major depression and anxiety) are more common in females. A goal of this research program is to investigate the biological basis for these sex differences in fetal mouse brain exposed to environmental stressors during early gestation, prior to the appear- ance of sex hormones. Published work by the PI has identified VPA-induced sex differences in the activating epigenetic mark, H3K4me3, leading to sexually-dimorphic expression of Bdnf, the gene encoding brain-derived neurotrophic factor. These studies with Bdnf establish an experimental paradigm for identifying other sexually- dimorphic proteins that regulate brain development and that may mediate the pathogenic effects of environ- mental stressors on brain development. An important clue to the underlying mechanism is that the enzymes that regulate H3K4me3, the H3K4-demethylases, JARID1C and JARID1D, are encoded by genes on the X and Y-chromosomes, respectively, and are therefore postulated to be differentially expressed in the two sexes. The specific aims of this research program are to 1) identify genes involved in early brain development (in addition to Bdnf) that are expressed differently in males and females in response to VPA due to sexually dimorphic H3K4 trimethylation and 2) test the hypothesis that JARID1 gene expression and enzyme activity are greater in males than in females, thereby providing a plausible mechanism for sexually dimorphic gene expression in the fetal brain. Identification of such genes, particularly if found to be associated with one or more developmentally relevant signaling pathways, will lead to a clearer understanding of the etiology of neurodevelopmental disor- ders such as ASDs and provide the basis for future, hypothesis driven initiatives to determine the pathogenic mechanisms of action of other environmental stressors acting during early gestation.