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II: Research Papers

CHAPTER 8

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,

E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it (R. Levy)

ABSTRACT

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.

INTRODUCTION

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 [1] 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 [7] 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

  • 1. For the AD mouse model Ab 1-42 brain infusion (alone or together with the antisense or the sense) to C57B1/6 mice was done using a microcosmic pump attached to cannula stereotaxically implanted into the right lateral cerebral ventricle. Mice behavioral patterns were detected using Y-maze.
  • 2. For the ALS mouse model, B6.Cg-Tg(SOD 1G93A) 1 Gur/J hemizy- gous transgenic male mice were obtained from Jackson Laboratoiy (Bar Harbor, ME, U.S.A). ARotarod (Rotamex-5, Columbus Instruments, Columbus, OH, USA) test was used to evaluate the motor performance of the mice using an accelerating paradigm of 0.12 rpm/s.
  • 3. Antisense oligonucleotide against cPLA,a were engineered using the computer-based approach RNADraw Vl.l (Mazura Multi- media, Stockholm, Sweden). An oligo-deoxynucleotide antisense (tcaaaggtctcattccaca) and its corresponding sense with phosphoro- thioate modifications on the last three bases at both 5’ and 3’ ends were used as described in our previous article [4]. Mice (around 25 g weight) received 10 pg/day phospho oligo-deoxynucleotides diluted in saline.
  • 4. Microglia were isolated from brains of mice C57BL 1 day old pups and grown in DMEM-F12 medium (10% FCS, 1% non-essential amino-acids, 11.4 pM (3-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin and 12.5 U/ml nystatin into poly-L-lysine coated flasks and kept at 37°C in a humidified atmosphere of 5% CO, (Figure 8.1).
Onset of pathology in Ab brain infusion mouse model dependent on elevated cPLA,a. Each group contained 10 mice. **p

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.

RESULTS

  • 1. Behavioral deficit was detected by reduction in spontaneous behavioral alterations using a Y-maze analysis at 8 weeks of Amyloid betaw, (Ap) brain infusion. Prevention of elevated cPLA,a protein expression by brain infusion of AS with Ap, prevented the behavioral deficit in comparison with none treated mice or mice treated with sense. Brain infusion of AS alone did not have a similar effect on mice behavior analyzed by Y-maze.
  • 2. To detennine the role of cPLA,a in the progression of the disease, 10 pg AS or sense were injected to the spinal cord one a week from week 15 (shortly before the onset of motor neuronal dysfunction) for 6 weeks. As shown in Figure 8.2, AS treatment prolonged survival in mice by 14 days (<0.05) and significantly delayed the onset of motor neuron dysfunction by 3 weeks. There was a significant (p<0.001) difference between the two groups.
  • 3. In order to determine whether the reduction in glia activation determined by CD40 expression in AS treated hmSODl mice is due to the reduction of inflammation in the spinal cord or due to regulation by cPLA,a, primary brain rat microglia were studied. As shown in Figure 8.3, addition of 10 ng/ml IFNy to primary microglia for 24 h caused a significant (p<0.0001) elevation of cPLA,a and of CD40 protein expression, as shown in the double immuno-fluoresce staining analysis. Preventing cPLA2a up-regulation by addition of 2pM AS 24 h prior to addition of IFNy prevented CD40 protein induction. Incubation with the corresponding sense that had no effect on the elevation of cPLA,a protein expression by either of the inducers, did not affect the elevation of CD40 protein expression.
AS Brain infusion to hmSODl mice shortly before the onset of motor neuron, dysfunction delayed the development of the disease

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.

Elevated CD40 expression by IFNy in primary microglia cells is regulated by cPLA,a

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.

DISCUSSION

Inhibition of cPLA,a upregulation in the cortex of Ab brain infusion (a mouse model of AD) as shown in our previous study [8] 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 [9] 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.

KEYWORDS

  • antisense drug treatment
  • cyclooxygenase-2
  • double immuno-fluoresce staining analysis
  • inducible nitric oxide synthase
  • microglia
  • pro-inflammatory enzymes

REFERENCES

  • 1. Stephenson, D. T., Lemere, C. A., Selkoe, D. J., & Clemens, J. A., (1996). Cytosolic phospholipase A2 (cPLA2) immune reactivity is elevated in Alzheimer’s disease brain. Neurobiol. Dis., 3, 51-63.
  • 2. Clemens, J. A., et al., (1996). Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat. Stroke, 27,527-535.
  • 3. Stephenson, D., Rash, K., Smalstig, B., Roberts, E., Johnstone, E., Sharp, J., Panetta, J., et al., (1999). Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration. Glia, 27,110-128.
  • 4. Raichel, L., Berger, Y., ICachko, L., Hadad, N., Karter, M., Solodkin, I., Williams, I. R., et al., (2008). Reduction of cPLA,a over expression-ап efficient anti-inflammatory therapy for collagen induced arthritis. Eur. J. Immunol., 38,1-12.
  • 5. Hadad, N., Burgazliev, O., Elgazar-Carmon, V., Solomonov, Y., Wueest, S., Item, F., Konrad, D., et al., (2013). Induction of cytosolic phospholipase A,a is required for adipose neutrophil infiltration and hepatic insulin resistance early in the course of high fat feeding. Diabetes, 62, 3053-3063.
  • 6. Rosengarten, M., Hadad, N., Solomonov, Y., Lamprecht, S., & Levy, R., (2016). Cytosolic phospholipase A,a has a crucial role in the pathogenesis of DSS-induced colitis in mice. Eur. J. Immunol, 46, 400-408.
  • 7. Shibata, N., Kakita,A., Takahashi, H., Ihara, Y., Nobukuni, K., Fujimura, H., Sakoda, S., & Kobayashi, M., (2010). Increased expression and activation of cytosolic phospholipase A, in the spinal cord of patients with sporadic amyotrophic lateral sclerosis. Acta Neuropathol, 119, 345-354.
  • 8. Sagy-Bross, C., Kasianov, K., Solomonov, Y., Braiman, A., Friedman, A., Hadad, N., & Levy, R., (2015). The role of cytosolic phospholipase A,a in Amyloid Precursor Protein upregulation induced by amyloid beta^-implication for neurodegeneration. J. Neurochem., 732,559-571.
  • 9. Solomonov, Y., Hadad, N., & Levy, R., (2016). Reduction of cytosolic phospholipase A,a up regulation delays the onset of symptoms in SOD1G93A mouse model of amyotrophic lateral sclerosis. J. Neuroinflammation, 13,134-146.
 
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