Gregory Brewer, PhD - US grants

Membrane morphogenesis is a universal aspect of biological cell division, intracellular organellogenesis and mult cellular development. We wish to demonstrate how specific proteins cause shape changes in biological membranes. Bacteriophage PM2 is a useful model because of its simple lipid bilayer membrane. Proteins coded by bacteriophage PM2 cause a bud to form in the membrane of its host, Alteromonas espejiana. We hope to describe the molecular aspects of this lipid-protein interaction by isolation of the gene(s) responsible for synthesis of a morphogenic protein(s). We have demonstrated coupled transcription-translation directed by restriction endonuclease fragments of PM2 DNA. One of these fragments codes for the synthesis of sp6.6, a viral structural protein implicated in membrane morphogenesis. This sp6.6 restriction fragment and others will be inserted into plasmid pBR322 for amplification in E. coli. In transformed cells, we hope to see extreme membrane morphogenesis caused by overproduction of sp6.6. We will also determine the amino acid sequence of sp6.6 by sequencing its DNA restriction fragment. Stretches of hydrophobic amino acids will suggest a model for lipid-protein interaction. The model will be tested for the effect on membrane morphogenesis of site-specific mutagenesis of the sp6.6 gene. These fragments will also be subjected to in vitro protein synthesis in the presence of membranes to determine whether the membrane morphogenic protein is inserted co-translationally or post-translationally. Thus, we expect to be able to describe the genetic, biochemical and physical principles of membrane morphogenesis.

The compounds proposed in this study will be evaluated as magnetic and spectroscopic mimics of the strongly magnetically coupled Fe (III)- Cu(II) binuclear pair of the terminal respiratory enzyme cytochrome c oxidase in the belief that such investigations will further our understanding of this enzyme. Although the complete structure of this enzyme is unknown, it is known that a heme iron (III) is magnetically coupled to a copper(II) through a bridging ligand which could be an imidazolate or thiolate. It is reasonable to investigate the magnetic properties of this system by examining simple complexes which contain the salient features of the enzyme. The experiments proposed in this work will examine two important properties of iron porphyrins which are relevant to the understanding of heme systems in general and to cytochrome c oxidase in particular. The first is the spin state of five coordinate iron (II) porphyrin monoadducts with an imidazole donor. The second is the extent of imidazolate mediated magnetic coupling which can occur between an iron atom in a porphyrin environment and a paramagnetic copper(II). The approach used here will be to design a series of metal chelates (M=Cu(II) or Ni(II)) which contain a covalently bound imidazolate. These complexes, when reacted with iron porphyrins, will form axial adducts as do other imidazole derivatives. Steric restrictions placed on the metal imidazolate complex will insure formation of monoadducts which will be binuclear imidazolate bridged complexes of iron porphyrins. Experiments conducted on these complexes will probe the spin state and magnetic coupling properties of iron(II) and iron(III) compounds. The nickel complexes will serve as diamagnetic blanks for the copper ones. In this manner the spin state of the iron atom can be investigated from the Fe-Im-Ni complexes and the magnetic coupling properties with the Fe-Im-Cu ones. The determination of spin state will be based on Electron Spin Rsonance, Mossbauer, and magnetic susceptibility experiments and the magnetic coupling on variable temperature magnetic susceptibility experiments. Full structural characterization of these complexes will allow for a detailed magnetostructural correlation of this type of iron porphyrin. The results will be used to interpret the magnetic properties of cytochrome c oxidase with an emphasis on structural implications.

We propose acquisition of a Hitachi H-7000 transmission electron microscope (TEM). Due to efficiencies in this state-of-the-art instrument system, it will replace two TEM's in the SIUSM EM Facility which are 12 and 15 years old. The TEM instrument system will be used by 7 NIH support investigators and provide opportunities for non-funded investigators to generate preliminary results for subsequent NIH application. Brewer will the new instrument for four projects: 1) to localize the binding site of an undeca-gold-labeled antibody that blocks transjunctional membrane conductance in lens membranes, 2) to quantitate the morphology of cytoplasmic vesicles in E. coli which are produce by cloned genes, 3) to describe the development of synapses in culture hippocampal neurons and 4) to detect paired helical filaments and aluminum toxicity in cultured neurons treated with serum from Alzheimer patients. Caspary will examine the age-related loss of hearing by analysis of the age-related loss of GABA-immunoreactive neurons in the superior olivary complex, the cochlear nucleus and the inferior colliculus. Cooper will study specific surface antigens of the STD pathogens Clamydia trachomatis and Neisseria gonorrhoeae and their interactions with human fallopian tube mucosa in organ culture. Lee will determine the transmitter content in sympathetic nerve terminals of cerebral blood vessels. Rybak will use morphometric to determine their effects on transmembrane ion distributions. Tewari will use the Hitachi H-7000 to examine the interactions of Histoplasma capsulatum with immune and non-immune lymphoid cells and human endothelial cells in culture. Walsh will continue to correlate synaptic morphology with auditory function during development of the cochlear nucleus. Amankwah will extend his previous studies on chronic maternal insults to the fetus in utero to the effects of maternal stress on morphological characteristics of fetal sciatic nerve. To continue to be on the forefront of morphological investigation, this substantial user group needs the efficiencies of a state-of-the-art electron microscope.

DESCRIPTION (from the applicant's abstract) The most significant etiological factor in Alzheimer's disease (AD) is age. But how does age contribute to the cellular and molecular pathology of AD? It is hypothesized that hippocampal neurons from aged animals are more susceptible to Abeta than younger neurons due to a decreased ability to generate the energy needed to control ion fluxes in this region of the AD brain. Novel techniques for the culture of aged neurons in serum-free medium have been developed recently by the PI, enabling examination of age-related cellular responses. New data have been obtained that subtoxic levels of lactate acidosis alter APP processing resulting in deposition of more amyloidogenic fragments. Also, neurons from aged rats are more susceptible to Abeta, lactate acidosis, and glutamate toxicity and fail to increase ATP levels to meet demands of increased ionic fluxes. Using neurons cultured from adult and aged rats, the first aim is to compare responses to fibrillar Abeta in terms of intracellular calcium, pH, and viability. Also regional AD pathology will be examined by comparing responses to Abeta in hippocampal with cerebellar granule neurons. The second aim examines the impact of glutamate and lactate acidosis on Abeta toxicity. The third aim examines whether age-related changes in susceptibility to Abeta are accounted for by declines in energy production, free radical damage, or induction of apoptosis. These studies hope to provide a better understanding of the age-related responses of cultured neurons to stressors thought to be important in the AD brain.

[unreadable] DESCRIPTION (provided by applicant): Age-Dependent Response of Hippocampal Neurons to Stress Age remains the most significant unexplained etiologic factor in common neurodegenerative diseases. To explore the basis of age-related sensitivity to common stressors, Ab and glutamate, we have developed novel techniques for isolation and culture of hippocampal rat neurons of any age (Brewer, 1997). Compared to embryonic and middle age neurons, we find that old neurons are more sensitive to glutamate and Ab toxicity (Brewer, 1998). Part of the mechanism involves age-related increases in apoptosis, indicated by more condensed nuclei and higher levels of caspase-3 activation. Here, we will test the hypothesis that age-related functional deficits in hippocampal mitochondria are responsible for age-related neurotoxicity of Ab and glutamate. Our aims will continue to use hippocampal neurons isolated from rat brains of three ages embryonic, middle and old age) to compare single cell function under uniform culture conditions: 1) Are age related functional deficits of mitochondria responsible for the age-related increase in susceptibility to glutamate and A-beta stressors? Mitochondrial deficits will be measured as respiration, steps in oxidative phosphorylation, preconditioning, release of cytochrome C and oxyradical products. 2) Can multiple inhibitor: better protect against age-related stressor toxicity than either alone? 3) Are there age-related deficits in mitochondrial DNA of mice transgenic for mutant human APP in situ hybridization with probes for common deletions? 4) Can neuron multiplication select for wild-type mitochondrial DNA and thereby restore normal function including response to stressors? These studies are likely to reveal mechanistic insights into age related neurotoxicity and suggest new targets for therapeutic intervention at both preventative and restorative levels of neurodegeneration.

Age-related Response of Hippocampal Neurons to Stress P.I.: Brewer, Gregory J. ABSTRACT Age remains the most significant unexplained etiologic factor in common neurodegenerative diseases. The issue of age is brought into sharper focus by the question, what is it about aging that causes people with congenital mutations in APP or presenilin to wait decades before invariably developing Alzheimer disease (AD) with cognitive deficits and brain pathology? Our previous NIA grant began to investigate the aging component for neurons from aging (24 mo.) compared to middle-age rats (9 mo.). The results firmly established the higher intrinsic susceptibility of old rat neurons to stressors such as glutamate and A[unreadable], particularly in mitochondrial function and bioenergetics. These changes must be intrinsic to old neurons because they remain in common culture conditions after removal from the aging hormone, immune and vasculature system environments. Our data suggest explicitly enhanced resting mitochondrial production of reactive oxygen species (ROS), low glutathione (GSH) and NADH and depolarized mitochondrial membrane potential (MMP). Other parameters such as viability in the common culture medium, mitochondrial number per cell and resting respiration are not different. Most importantly, most of the impairments are only evident after treatment with glutamate or A[unreadable]. While evidence for age-related damage to nucleic acids, proteins and lipids abound, the proposed studies will strategically focus on the sources or conditions that promote this damage. Specifically, it is proposed that aging increases susceptibility to stressors like glutamate and A[unreadable] by an incremental failure of the essential signaling functions of ROS redox control. Since energy supply is so critical to synaptic function, Aim 1 will determine the cause of the age-related depolarization of the MMP and higher levels of ROS in old rat neurons and also examine inefficient autophagy in old neurons due to lower turnover of mitochondria. Aim 2 will investigate the consequences to the aging oxidizable proteome of this age-related loss of neuronal GSH, NADH and redox potential. As the final stage of epigenetic development, Aim 3 will examine the epigenetic controls for this age-related increase in susceptibility to stressor toxicity in old rats, including histone acetylation and CpG methylation of mitochondrial promoters. Since estrogen is neuroprotective to old neurons at pM concentrations, the ability of estrogen to reverse the changes observed in each aim will also be determined. Completion of the proposed studies should provide definitive evidence for a mechanistic basis of age-related stressor susceptibility and nutraceutical intervention.

DESCRIPTION (provided by applicant): Our goal is to establish a mechanism for failure of aging brain mitochondria to produce enough energy during stress, mitoenergetic failure. Without stress, we have recent evidence that mitochondria from old rat neurons in culture promote normal neuron survival, normal regeneration, normal glucose uptake and normal respiration, but do so with decrepit mitochondria which fail to upregulate energy production under stress. Compared to middle-age neurons, old neuron mitochondria are considerably depolarized, produce higher levels of ROS with lower glutathione antioxidant and maintain a more oxidized redox potential [ NAD(P)H / FAD ], all of which contribute to increased susceptibility to toxic stressors such as glutamate and beta-amyloid. Now we will use a well-established model of Alzheimer disease, LaFerla's 3xTg-AD mouse to test our hypothesis that redox potential and mitoenergetic function are impaired early in 3xTg-AD mice, compared to wild-type mice of the same age, but similar to old wild-type mice. More specifically, we hypothesize that an oxidized redox potential develops early during aging and causes a mitochondrial metabolic shift to begin a vicious cycle of insulin resistance, inhibited mitochondrial turnover and slothful energetics all of which are enforced by epigenetic mechanisms. To focus on the intrinsic neuronal differences with age, isolated from hormonal, vascular and immunologic aging, we will continue to culture neurons from these adult mice in a common culture condition. Neuron cultures also provide greater power for larger samples during exposure to varying concentrations of glutamate stress. In this mouse model of AD, Aim 1 will establish upstream causes of mitochondrial dysfunction as more oxidized redox potential and depolarized mitochondria, an age-related increase in ROS and lower glutathione. Aim 2 will evaluate a mechanistic basis for mitoenergetic failure as an age-related increase in insulin signaling and decrease in transcriptional activators in the peroxisome proliferator family, especially PGC1a, PPAR? and NRF, also known to stimulate mitochondrial biogenesis. Since changes in mitochondrial function with age persist in culture, in Aim 3, we will test the hypothesis that premature aging of mitochondria is controlled by either a) the efficiency of autophagy for turnover of decrepit mitochondria or b) epigenetic controls of histone acetylation and CpG methylation. This work is a collaboration between an aging mitochondria and neuron culture expert, Greg Brewer, a mouse aging expert, Andrzej Bartke and an insulin signaling transcription aging expert, Michal Masternak, all at Southern Illinois University School of Medicine. Overall, completion of these experiments should provide definitive mechanistic evidence for age-related control of mitochondrial energetics and insulin sensitivity critical for resistance to stressors with age. PUBLIC HEALTH RELEVANCE: The overall goal of the proposed work is to determine the mechanism of action of aging within mitochondria of neurons in the aging brain. Once specific targets are identified, then appropriate behavioral, nutraceutical or pharmacological proscriptions can be developed. These issues are critical to the aging population in the U.S. who will constitute 23% of the population in 2040, 70 million people. 13 million of these people are expected to have Alzheimer disease with expected health care costs of $250 billion.