Devin S. Gary

Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor.

Ischemic and excitotoxic insults to the brain induce rapid production of tumor necrosis factor-α (TNF), but the role of TNF in neuronal responses to brain injury are unclear. Two different TNF receptors (p55 and p75) are expressed in neurons and glia, To understand the role of TNF in brain injury, we generated mice that lack p55, p75, or both receptors, We report that neuronal damage after focal cerebral ischemia—reperfusion is significantly increased in mice lacking p55 receptors (85 ± 7 mm3 infarct volume; mean ± SD) compared with wild-type mice (70 ± 8 mm3) and mice lacking p75 receptors (72 ± 6 mm3). Moreover, mice lacking p55 receptors exhibited increased degeneration of CA3 hippocampal neurons after administration of the excitotoxin kainic acid compared with wild-type mice and mice lacking p75 receptors. When taken together with recent data showing that TNF can prevent apoptosis of cultured neurons exposed to oxidative and metabolic insults, our findings suggest that TNF plays a neuroprotective role after acute brain insults.

Integrin signaling via the PI3-kinase-Akt pathway increases neuronal resistance to glutamate-induced apoptosis

Integrins are integral membrane proteins that mediate adhesive interactions of cells with the extracellular matrix and with other cells. Integrin engagement results in activation of intracellular signaling cascades that effect several different cellular responses including motility, proliferation and survival. Although integrins are known to provide cell survival signaling in various types of non‐neuronal cells, the possibility that integrins modulate neuron survival has not been explored. We now report data demonstrating a neuroprotective function of integrins in embryonic hippocampal neurons. Neurons grown on laminin, an integrin ligand, exhibit increased resistance to glutamate‐induced apoptosis compared with neurons grown on polylysine. Neurons expressed integrin β1 and treatment of cultures with an antibody against integrin β1 abolished the protective effect of laminin. Neurons maintained on laminin exhibited a sustained activation of the Akt signaling pathway demonstrated in immunoblot analyses using an antibody that selectively recognizes phosphorylated Akt. The neuroprotective effect of integrin engagement by laminin was mimicked by an IKLLI‐containing integrin‐binding peptide and was abolished by treatment of neurons with the PI3 kinase inhibitor wortmanin. Levels of the anti‐apoptotic protein Bcl‐2 were increased in neurons grown on laminin and decreased by wortmanin, suggesting a mechanism for the neuroprotective effect of integrin‐mediated signaling. The ability of integrin‐mediated signaling to prevent glutamate‐induced apoptosis suggests a mechanism whereby neuron–substrate interactions can promote neuron survival under conditions of glutamate receptor overactivation.

4-hydroxynonenal, a product of lipid peroxidation, damages cholinergic neurons and impairs visuospatial memory in rats

The mechanisms that underlie cholinergic neuronal degeneration in Alzheimer disease (AD) are unclear, but recent data suggest that oxidative stress plays a role. We report that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, damages and kills basal forebrain cholinergic neurons when administered intraparenchymally. Examination of Nisslstained brain sections following unilateral HNE infusion revealed widespread neuronal loss in basal forebrain ipsilateral to the injection, but not on the contralateral side. Levels of choline acetyltransferase activity and immunoreactivity in the ipsilateral basal forebrain and hippocampus were significantly reduced by 60-80% seven days following HNE administration. Performance in Morris water maze tasks of visuospatial memory was severely impaired in a dose-dependent manner seven days following bilateral administration of HNE. Bilateral infusion of FeCl2 (an inducer of membrane lipid peroxidation) into the basal forebrain caused neuron loss and decreased choline acetyltransferease immunoreactivity and deficits in visuospatial memory. Additionally, FeCl2 infusion increased HNE immunoreactivity, implicating HNE in iron-induced oxidative damage. Because recent studies have demonstrated HNE adducts in degenerating neurons in AD brain, the present findings suggest a role for HNE in damage to cholinergic neurons in AD.

RNA Interference in Biology and Medicine

First discovered in plants the nematode Caenorhabditis elegans, the production of small interfering RNAs (siRNAs) that bind to and induce the degradation of specific endogenous mRNAs is now recognized as a mechanism that is widely employed by eukaryotic cells to inhibit protein production at a post-transcriptional level. The endogenous siRNAs are typically 19- to 23-base double-stranded RNA oligonucleotides, produced from much larger RNAs that upon binding to target mRNAs recruit RNases to a protein complexthat degrades the targeted mRNA. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. RNA interference methods provide several major advantages over prior methods (antisense DNA or antibody-based techniques) for suppressing gene expression. Recent preclinical studies suggest that RNA interference technology holds promise for the treatment of various diseases. Pharmacologists have long dreamed of the ability to selectively antagonize or eliminate the function of individual proteins—RNAi technology may eventually make that dream a reality.

Neuroprotective and neurorestorative signal transduction mechanisms in brain aging: modification by genes, diet and behavior.

Cells in the brain deploy multiple mechanisms to maintain the integrity of nerve cell circuits, and to facilitate responses to environmental demands and promote recovery of function after injury. The mechanisms include production of neurotrophic factors and cytokines, expression of various cell survival-promoting proteins (e.g. protein chaperones, antioxidant enzymes, Bcl-2 and inhibitor of apoptosis proteins), protection of the genome by telomerase and DNA repair proteins, and mobilization of neural stem cells to replace damaged neurons and glia. The aging process challenges such neuroprotective and neurorestorative mechanisms, often with devastating consequences as in Alzheimers disease (AD), Parkinsons and Huntingtons diseases and stroke. Genetic and environmental factors superimposed upon the aging process can determine whether brain aging is successful or unsuccessful. Mutations in genes that cause inherited forms of AD (amyloid precursor protein (APP) and presenilins), Parkinsons disease (alpha-synuclein and parkin) and trinucleotide repeat disorders (e.g. huntingtin and the androgen receptor) overwhelm endogenous neuroprotective mechanisms. On the other hand, neuroprotective mechanisms can be bolstered by dietary (caloric restriction, and folate and antioxidant supplementation) and behavioral (cognitive and physical activities) modifications. At the cellular and molecular levels, successful brain aging can be facilitated by activating a hormesis response to which neurons respond by upregulating the expression of neurotrophic factors and stress proteins. Neural stem cells that reside in the adult brain are also responsive to environmental demands, and appear capable of replacing lost or dysfunctional neurons and glial cells, perhaps even in the aging brain. The recent application of modem methods of molecular and cellular biology to the problem of brain aging is revealing a remarkable capacity within brain cells for adaptation to aging and resistance to disease.

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin‐linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the α chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a β1 integrin‐dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin‐activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide‐3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor‐induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin‐mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate‐associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.

PTEN regulates Akt kinase activity in hippocampal neurons and increases their sensitivity to glutamate and apoptosis.

The tumor suppressor phosphatase PTEN can promote apoptosis of mitotic cells by inhibiting activation of the cell survival kinase Akt. PTEN is essential for normal embryonic development, PTEN expression is associated with neuronal differentiation, and deletion of PTEN in the mouse brain results in seizures, ataxia, and other abnormalities. However, the possible roles of PTEN in regulating neuronal survival are not known. We provide evidence that PTEN sensitizes hippocampal neurons to excitotoxic death in culture and in vivo. Overexpression of wild-type PTEN decreased, while a dominant-negative PTEN increased, levels of activated Akt in cultured hippocampal neurons. Wild-type PTEN promoted, while dominant-negative PTEN prevented, apoptotic death of neurons exposed to the excitatory amino acid neurotransmitter glutamate. Hippocampal neurons of mice with reduced PTEN levels were more resistant to seizure-induced death compared to wild-type littermates. These findings demonstrate a cell death function of PTEN in hippocampal neurons and identify PTEN as a potential therapeutic target for neurodegenerative disorders that involve excitotoxicity and apoptosis. The ability of PTEN to modify neuronal sensitivity to glutamate also suggests possible roles for PTEN in regulating developmental and synaptic plasticity.

Involvement of Gadd153 in the pathogenic action of presenilin‐1 mutations

Mutations in the presenilin‐1 (PS1) gene cause early onset familial Alzheimers disease (FAD) by a mechanism believed to involve perturbed endoplasmic reticulum (ER) function and altered proteolytic processing of the amyloid precursor protein. We investigated the molecular mechanisms underlying cell death and ER dysfunction in cultured cells and knock‐in mice expressing FAD PS1 mutations. We report that PS1 mutations cause a marked increase in basal protein levels of the pro‐apoptotic transcription factor Gadd153. PS1 mutations increase Gadd153 protein translation without affecting mRNA levels, while decreasing levels of the anti‐apoptotic protein Bcl‐2. Moreover, an exaggerated Gadd153 response to stress induced by ER stress agents was observed in PS1 mutant cells. Cell death in response to ER stress is enhanced by PS1 mutations, and this endangering effect is attenuated by anti‐sense‐mediated suppression of Gadd153 production. An abnormality in the translational regulation of Gadd153 may sensitize cells to the detrimental effects of ER stress and contribute to the pathogenic actions of PS1 mutations in FAD.

Concentration- and cell type-specific effects of calbindin D28k on vulnerability of hippocampal neurons to seizure-induced injury

The calcium-binding protein calbindin D28k (CB) is expressed in limited subpopulations of neurons in the brain. In the hippocampus, CB is expressed in all dentate granule cells and a subpopulation of CA1 pyramidal neurons, but is absent from CA3 neurons. This pattern of CB expression is inversely correlated with neuronal vulnerability to seizure-induced damage suggesting the possibility that expression of CB confers resistance to excitotoxicity. While data from cell culture studies support an excitoprotective role for calbindin, it is not known whether CB is a key determinant of neuronal vulnerability in vivo. We therefore examined the pattern of damage to hippocampal neurons following intrahippocampal injection of the seizure-inducing excitotoxin kainate in CB homozygous (CB-/-) and CB heterozygous (CB+/-) knockout mice in comparison with wild-type mice (CB+/+). Whereas the extent of damage to CA1 neurons was similar in CB-/- and CB+/+ mice, damage to CA1 neurons was significantly reduced in CB+/- mice. Dentate granule neurons were not damaged following kainate-induced seizures in CB+/+, CB+/- or CB-/- mice. These findings suggest that CB can modify vulnerability of hippocampal CA1 neurons to seizure-induced injury, and that either CB is not a critical determinant of resistance of dentate granule neurons, or compensatory changes occur and lack of CB is not the only difference between CB-/- and CB+/+ mice.

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non‐ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP‐1 coimmunoprecipitates with the α2β1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP‐1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Giα containing heterotrimers. As an example, α2β1 can form a complex with CD47 that may associate with Giα. In the present study we have therefore investigated the possibility that MMP‐1 may affect intracellular changes that are linked to the activation of a Gi protein‐coupled receptor. We show that treatment of neural cells with MMP‐1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP‐1 potentiates proteinase activated receptor‐1 (PAR‐1) agonist‐linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein‐coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP‐1 stimulates pertussis toxin sensitive release ofMMP‐9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP‐1 signals through a pertussis toxin‐sensitive G protein‐coupled receptor.