Nucleic Acid Aptamer Selection Against Interleukin (IL-1β), Catabolin, to Analyze and Diagnose Chronic Neuropathic Pain Patients

Nucleic Acid Aptamer Selection Against Interleukin (IL-1β) to Analyze and Diagnose Chronic Neuropathic Pain Patients

Gabbe Zuniga

16 October 2012

Abstract –

      The evaluation and treatment of chronic neuropathic pain is controversial.  Doctors continue to question whether the transition from acute to chronic pain is due to pain sensitivity from injury or associative learning from general stress.  It has been proposed that hippocampal-cortical learning processes play an important role in the development and maintenance of chronic neuropathic pain from nerve injury (1).

Abnormal hippocampal function, such as decreased neurogenesis and reduced kinase expression and phosphorylation, due to neuropathic pain has been found to be associated with cytokine gene expression (2).  Interleukin-1β, a proinflammatory cytokine, plays a role in brain function during inflammatory responses by influencing synaptic plasticity and memory processes in the hippocampus (3).  IL-1β is targeted to many different cell types involved in the central nervous system (CNS) in which its role is to initiate activation of inflammatory mediators, such as proinflammatory cytokines and neutrophil-recruiting chemokines, in response to CNS injury (4).  As shown in Figure 1, IL-1β is responsible for multiple complex intracellular signaling pathways that are correlated with impairment of long-term potentiation (LTP), which is responsible for enhancing signal transmission and therefore synaptic strength (5).

Based on IL-1β biological effects, localization and competitive inhibition of IL-1β can prove useful in clinical diagnostic and therapy for chronic pain patients.  The development of high-affinity nucleic acids, also called aptamers, as molecular recognition and binding elements to IL-1β can serve as therapeutic agents.  An aptamer might be able to prevent leukocyte migration and activation of astrocytes as well as microglia, both of which are important components to the brain’s innate immune response.  IL-1β binding studies have shown that the presence of IL-1 receptors (IL-1R) in the brain is highest in the molecular and granular layers of the dentate gyrus of the hippocampus (3)(6).  In order to reaffirm these results and localize expression, a green fluorescent protein marker bound to the aptamer will allow its progress to be tracked.  Inhibition of the binding mechanism between IL-1β and IL-1R has been shown to decrease the maintenance of LTP by inhibiting adequate Ca2+ influx required to initiate biochemical cascades for LTP and increasing circulating corticosteroids as well as other stress-induced mechanism changes (5)(7).  Ultimately, this will affect learning and memory of pain stimuli, which will allow for a better understanding of the mechanisms of localized and widespread neuropathic pain. 

Specific Aim 1 –

Selection of aptamer against IL-1β.

An aptamer for IL-1β that will inhibit the protein may result in the following changes in the nervous system (i.e., hippocampus).  Inhibition will decrease the maintenance of long-term potentiation, thus, affecting learning and memory of pain stimuli.  This will potentially minimize the psychological stress factor associated with pain and lead to a more accurate diagnosis of patients; and therefore, improve the applied therapeutic treatments.  An aptamer interacting with IL-1β will block activation of other proteins involved in IL-1 signal transduction of the neuroimmune pathway.  The mechanisms by which IL-1β affects the hippocampal LTP are shown in Figure 1.

This function can be applied to further studies evaluating whether the IL-1β and IL-1R polymorphisms are also involved in induced neuropathic pain in the musculoskeletal systems similar to the nervous system (i.e., hippocampus).  If the results (symptoms) of non-neuronal (joints, ligaments, etc.) and neuronal (N-methyl-D-aspartic acid (NMDA) receptor antagonists, opioid analgesics, and sodium channel blockers) targets after inhibition of IL-1β are similar to the previous studies, then it could be concluded that IL-1β plays an equally important part in chronic pain development in both neuropathic pain and neurodegenerative diseases (7).

Specific Aim 2 –

Develop fluorescent aptamer marker to detect IL-1β localization.

The specific aim is to develop an aptamer against IL-1β that is marked by a green fluorescent protein.  By doing so, the levels of IL-1β can be detected and localized in living cells by fluorescence microscopy.  This information can clarify where the highest density of IL-1β and IL-1R interaction occurs and therefore, locate the major site of the signaling cascade required for a sufficient immune response to chronic neuropathic pain.

            The green fluorescent protein can be tracked in animal models to allow their expression and localization to be examined in real time in living cells (8). The progress of IL-1β after induced nerve injury can be used to either support or reject the idea of using IL-1β as a target for interrupting the pathogenesis of chronic neuropathic pain (9).  If these results indicate that high densities of IL-1β transcription and expression are located in critical areas for learning and memory such as the CA1, CA3, and/or denate gyrus of the hippocampus, then it can be concluded that IL-1β-mediated inflammatory processes in the hippocampus play a role in memory impairment (5).    Figure 1. IL-1β is activated after cleavage by MCP-1 (capase-1).  Inhibition of IL-1β from signaling a series of intracellular signaling cascades will prevent transcription factor activation and gene expression, thus, decreasing the neuroinflammation immune response to the site of injury.  IL-1β also inhibits hippocampal long-term potentiation. By inhibiting IL-1β from binding to its corresponding receptors, IL-1R1 and IL-1R, in the hippocampus, maintenance of long-term potentiation will be improved.

Interleukin-1 beta Human Recombinant Protein can be ordered from Novus Biologicals, LLC online at novus@novusbio.com or by phone at 303.730.1950.  Its catalog number is NBC1-18478.  The protein is purchased at $295 for 0.1 mg (100 μg) at a concentration of 1.0 mg/mL (100 μg/100 μL) with no preservative.  It will cost $10.25 each round of SELEX to use ~200 pmol of target.      

 Figure 1. IL-1β is activated after cleavage by MCP-1 (capase-1).  Inhibition of IL-1β from signaling a series of intracellular signaling cascades will prevent transcription factor activation and gene expression, thus, decreasing the neuroinflammation immune response to the site of injury.  IL-1β also inhibits hippocampal long-term potentiation. By inhibiting IL-1β from binding to its corresponding receptors, IL-1R1 and IL-1R, in the hippocampus, maintenance of long-term potentiation will be improved.  

Product: IL-1β Human Recombinant Protein

Size: 100 μg

Daltons: 17,376 grams/mole

Website: Novus Biologicals (novus@novusbio.com)

Catalog #: NBC1-18478

Price: $295

Price per round: $10.25

References –

(1). O’Connor, John, and Andrew Coogan.  “Actions of the Pro-Inflammatory Cytokine IL-1β on Central Synaptic Transmission.”  Experimental Physiology 84 (1999): 601-614. Web. 27 Aug. 2012.

(2). Mutso, Amelia et al.  “Abnormalities in Hippocampal Functioning with Persistent Pain.”  The Journal of Neuroscience. 32.17 (2012): 5747-5756. Web. 4 Sept. 2012. 

(3). Gao, Yong-Jing and Ru-Rong Ji.  “Targeting Astrocyte Signaling for Chronic Pain.” Neurotherapeutics. 7.4 (2010): 482-493. Web. 27 Aug. 2012.

(4).  “Chronic IL-1 beta-induced Neuroinflammation: Is it Really that Bad?” (2012). R&D Systems Inc. Web. 27 Aug. 2012. 

(5). Lynch, M. A. “Long-Term Potentiation and Memory.” American Physiological Society.  (2004): n. pag. Web. 27 Aug. 2012. 

(6). Schneider, H. et al. “A neuromodulatory role of interleukin-1β in the hippocampus.” Proc. National Academic Science USA. 95 (1999): pp. 7778-7783. Web. 27 Aug. 2012.

(7). Shaftel, Solomon et al. “The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective.” Journal of Neuroinflammation.  5 (2008):7. Web. 27 Aug. 2012.

(8). Bruges, Jacome Armas et al. Update on Immunogenetics of Spondyloarthritis. New Research on Immunology. Veskler, Barbara A. New York City: Nova Science Publishers, Inc., 2005. Google eBook. Web. 27 Aug. 2012.

(9). Cibelli et al. “Role of Interleukin-1beta in postoperative cognitive dysfuncton.” Annals of Neurology. 68.3 (2010): 360-368. Web. 27 Aug. 2012. 

Continue to Final Target Proposal. 
Progress Report 1
Progress Report 2
Final Report

Nucleic Acid Aptamer Selection Against Tie-2 to Limit Tumor Growth

Amika Alibhai 

Link to Proposal

Link to Progress Report 1 

Link to Progress Report 2 

Link to Final Manuscript

Nucleic Acid Aptamer Selection Against Tie-2 to Limit Tumor Growth

Abstract

A tumor is a population of cancer cells, or cells that stop dividing in an orderly manner and start rapidly growing, dividing, and gaining mutations.  However, due to a lack of nourishment, lone tumors cannot grow past one or two cubic millimeters ⁽¹⁾.  In order to grow larger, tumors require nutrients, which are usually delivered though blood vessels.

Tie-2, a receptor tyrosine kinase, is an endothelial cell surface receptor that binds to angiopoietins and mediates cell signaling through phosphorylation and binding and activation of enzymes ⁽²⁾.  An angiopoietin is a protein growth factor required for angiogenesis, the formation of blood vessels.  Angiopoietins function by binding receptor tyrosine kinases, such as Tie-2, which is mandatory for normal embryonic vascular development and tumor angiogenesis ⁽³⁾.  While angiogenesis is vital in the healing of wounds, it also serves in the growth and transition of tumors from dormant to malignant, as well as contributing to metastasis.  Tie-2, by mediating the effects of angiopoietins, is an integral receptor in the pathway of blood vessel formation ⁽⁴⁾.

As tumors are nourished and led through metastasis, the process becomes more agonizing for cancer patients and doctors alike.  To manage this there are general antibody treatments available, but another method of therapy is possible.  Such a possibility comes in the form of an aptamer, which is a short sequence of nucleic acids that binds with great specificity and strength to a particular target.  Isolated in vitro from vast libraries of sequences, aptamers can fold into tight binding pockets for nearly any molecule ⁽⁵⁾.  Aptamers are expected to be able to bind Tie-2; an aptamer that blocks the binding site of angiopoietins would reduce signaling vital to the growth of tumors by angiogenesis.  In this respect, an aptamer would be beneficial in cancer treatment and therapy by limiting the progression of tumors in early stages.

Specific Aim 1:  RNA aptamer selection against Tie-2

RNA aptamers are selected through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process ⁽⁶⁾.  Tie-2 proteins are functionalized with 6X Histidine tags, lending to use of Nickel-NTA magnetic beads in the process of selection.

Specific Aim 2:  Develop an aptamer therapeutic to limit tumor growth

Upon the binding of the angiopoietins, Tie-2 provides the signaling essential to angiogenesis.  Tie-2 also mediates the actions of angiopoietins in their prevention of apoptosis, further aiding the growth of blood vessels ⁽⁷⁾.  An aptamer selected against Tie-2 may inhibit the receptor, thus suppressing the development and spreading of tumors by depriving the pathway of the required signaling.  

Figure 1.  The progression from Tie-2 to tumor growth.

A comparison is shown between an angiopoietin and an aptamer in binding to Tie-2.

Recombinant Human Tie-2 with a 6-His tag is available online from R&D Systems (rndsystems.com).  The catalog number is 313-TI-100 and the price is $340 for 100 micrograms.  The protein is 165 kDa and is to be resuspended at 100 ug/mL; this will provide six rounds of selection at approximately $56 per round, if using 100 pmol per round.

References

1. McDougall, S.R, Anderson, A.R.A., Chaplain, M.A.J., (2006) “Mathematical modelling of dynamic adaptive tumour-induced angiogenesis: Clinical implications and therapeutic targeting strategies.” Journal of Theoretical Biology. 241.

2. Cox, M., Nelson, D.R. (2008). Lehninger: Principles of Biochemistry (fifth ed.). W H Freeman & Co.

3. National Library of Medicine – Medical Subject Headings, (2011).  “Receptor, Tie-2.”  nlm.nih.gov. 

4. Fagiani, E., Christofori, G.,  (2012).  “Angiopoietins in Angiogenesis.”  Cancer Lett.  Ahead of print.

5. Famlouk, M., Hartig, J.S., Mayer, G., (2007). “Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy.”  Chem Rev. 107:3715-43.

6. Ellington, A.D. Szostak, J.W., (1990.) “In vitro selection of RNA molecules that bind specific ligands.” Nature. 346:818-822.

7. Milner, C.S., Hansen, T.M., Singh, H., Brindle, N.P., (2009).  “Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angipoietin-1 on endothelial permeability and apoptosis.” Microvasc Res. 77:187-91.