Table of Contents:
II: Research Papers
Elevated Cytosolic Phospholipase A2a as a Target for Treatment and Prevention the Progression of Neurodegenerative Diseases
RACHEL LEVY, YULIA SOLOMONOV, KESENIA KASIANOV,
YAFA MALADA-EDELSTEIN, and NURIT HADAD
Department of Clinical Biochemistry and Pharmacology,
Faculty of the Health Sciences Ben-Gurion University of the Negev and Soroka Medical University Center, BeerSheva, Israel,
Intravenous injections of an antisense against the main pro-inflammatory enzyme; cytosolic phospholipase A,a (cPLA,a) reduced cPLA,a upregulation specifically at the site of inflammation. To study the role of cPLA,a in neurodegenerative diseases a specific antisense against cPLA,a (AS) was brain infused to inhibit cPLA,a upregulation in the brain. Brain infusion of the antisense drug in a mouse model of amyloid brain infusion, representing a mouse model of Alzheimer’s disease (AD), was found to be efficacious in preventing cPLA,a upregulation in the brain and in the prevention of the disease. Reduction of the elevated expression of cPLA,a in the spinal cord of human SOD1G93A transgenic (hrnSODl) mice (a mouse model for amyotrophic lateral sclerosis (ALS)) by brain infusion of AS at week 15 (shortly before the appearance of the disease symptoms) for a duration of 6 weeks, delayed the loss of motor neuron function in comparison with limSODl mice and with sense brain infused hmSODl mice. Since specific reduction of cPLA,a in the brain and spinal cord significantly attenuated the development of the diseases in mouse models of AD and ALS, cPLA2a may offer an efficient target for treatment neurodegenerative diseases.
The role of inflammation in the pathogenesis of a vast array of diseases including neurodegenerative diseases has been well documented. Elevated cytosolic phospholipase A,a (cPLA,a) expression and activity were detected in the inflammatory sites in a vast array of inflammatory diseases, including neurodegenerative diseases [1, 2]. cPLA,a is a requisite component in the cascade of events leading to the production of eicosanoids during acute and chronic inflammation. Intravenous injections of an antisense against cPLA,a reduced cPLA,a upregulation specifically at the site of inflammation. Intravenous injections of the AS in mouse models of peritonitis, arthritis, colitis, and insulin resistance, proved efficacious for the treatment or prevention of these diseases [4-6]. Increased cPLA,a immunoreactivity and transcript were observed in Alzheimer’s disease (AD) in the brain  often associated with amyloid deposits, suggesting its role in the pathogenesis of the disease. Increased expression and activity of cPLA,a has been detected in neurons, astrocytes, and in microglia in the spinal cord, brainstem, and cortex of sporadic ALS patients  and in the spinal cord of G93A human mutant transgenic (hmSODl) mice, suggesting that cPLA,a may have an important role in the pathogenesis of the disease in all ALS patients.
MATERIALS AND METHODS
FIGURE 8.1 Onset of pathology in Ab brain infusion mouse model dependent on elevated cPLA,a. Each group contained 10 mice. **p<0.05 significant decrease in mice infused with Ab or Ab + sense compared with control mice (with buffer) or mice infused with AS + Ap.
FIGURE 8.2 AS Brain infusion to hmSODl mice shortly before the onset of motor neuron, dysfunction delayed the development of the disease. To the spinal cord of 15 weeks old hmSODl mice 10 ug AS or the corresponding sense were injected once a week during 6 weeks (n = 7 in each group). AS brain infusion prolonged survival and delayed loss of motor function analyzed by Rotarod */><0.001-significance relative to sense treated hmSODl mice.
FIGURE 8.3 Elevated CD40 expression by IFNy in primary microglia cells is regulated by cPLA,a. A representative double-immunofluorescence staining of cPLA,a (green) and CD40 (red) in unstimulated or stimulated microglia by 25 ng/ml IFNy in the absence or presence of 2 u.Vl AS or 2 u.Vl sense (SE). Scale bars = 50pm. The intensity of CD40 or cPLA,a were quantitated for cell and expressed in the bar graph as arbitrary units. ***p<0.0001- significance differences.
Inhibition of cPLA,a upregulation in the cortex of Ab brain infusion (a mouse model of AD) as shown in our previous study  prevented the behavioral deficit. Similarly, inhibition of cPLA,a upregulation in the spinal cord of limSODl mice (a mouse model of ALS) at the onset of the disease symptoms  significantly delayed the development of the disease. Increased expression and activity of cPLA,a have been detected in all cell types in the spinal cord, brainstem, and cortex of both sporadic and familial ALS, suggesting that cPLA,a may have an important role in the pathogenesis of the disease in all ALS patients. The antisense treatment that reduced cPLA,a upregulation in the brain and/or the spinal cord of antisense treated mice, prevented the reduction in the number of neurons (detected by NeuN), inhibited astrocyte activation (detected by GFAP) and microglial activation (detected by Iba-1 and/or by CD40). In addition, antisense treatment blunted the upregulation of the pro-inflammatory enzymes: inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The activation of microglia detected by CD40 overexpression is regulated by cPLA,a. In conclusion, antisense drug treatment is an exciting and emerging specialty area, not as yet in common use. Various antisense drugs for a variety of diseases and disorders are now in clinical phase testing, evincing the potential and promise of antisense drugs as a treatment strategy.