(L-444) Exploring the use of protamine as a therapeutic for the treatment of traumatic injury to the central nervous system

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row L - Poster # 444

Presenting Author(s)

DN

Derek Nelson

Ph.D. Student
Rensselaer Polytechnic Institute, United States

Co-Author(s)

JF

Jessica Funnell

Ph.D. Student
Rensselaer Polytechnic Institute, United States
CM

Christina Mendoza

Ph.D. Student
Rensselaer Polytechnic Institute, United States
GQ

Gerri Quinones

Senior Research Technician
Rensselaer Polytechnic Institute, United States
MB

Marvin Bentley

Assistant Professor
Rensselaer Polytechnic Institute, United States

Primary Investigator(s)

RG

Ryan Gilbert

Professor
Rensselaer Polytechnic Institute, United States

Introduction:: Traumatic injury to the central nervous system (CNS) results in multiple biochemical cascades that lead to the formation of the glial scar. While the glial scar does hinder secondary injury by stabilizing the injury site, it also provides a neuroinhibitory environment that prevents axonal regeneration and, ultimately, functional recovery. One of the major neuroinhibitory components of the glial scar is extracellular chondroitin sulfate proteoglycans (CSPGs), which are highly anionic components that play multiple roles in the nervous system [1]. Previous work shows that CSPG removal or sequestering can result in improved functional recovery after injury [2,3]. Sequestering these neuroinhibitory cues via small cationic therapeutics may provide a more immediate therapeutic response with a wider variety of delivery methods, compared to enzymatic or invasive treatments. While a previous study proposed the use of synthetic polypeptides to sequester these signals, their synthetic steps would significantly impact scale-up costs and they would need a full FDA investigation before being used in a clinical application [3]. However, there already exists a cationic polypeptide commonly used during surgeries. Protamine, an inexpensive and FDA-approved drug, is commonly used to reverse the anti-clotting activity of heparin during cardiac and vascular surgeries. However, its application for CNS injury, specifically its effect on neurons in neuroinhibitory/anionic environments, remains elusive. This study assesses the use of protamine for neural tissue injury concerning both neurons and glia. We hypothesized that protamine can be safely used to sequester neuroinhibitory signals and provide a new therapeutic for addressing traumatic CNS injury

Materials and Methods:: Rat neurons and mixed cortical glia (MCG) were used to assess protamine toxicity. Various concentrations of protamine (0.1 µg/mL – 1 mg/mL) were added to each primary cell type and subsequently cultured for 24 hours. Cell viability assays were used, including a lactate dehydrogenase (LDH) assay, calcein/Hoechst staining, and fluorescence analyses. Additionally, immunofluorescence was used to observe neuron adhesion and neurite outgrowth on inhibitory substrates with or without protamine. Lastly, the reactivity of astrocytes and their production of various CSPGs was studied in the presence of protamine using quantitative PCR methods.

Results, Conclusions, and Discussions:: The toxicity assays indicated that both neurons and MGCs showed the first signs of cell death at a protamine concentration of 10 µg/mL. Furthermore, protamine concentrations of 0.1 and 1µg/mL resulted in improved neuronal survival compared to the negative control that received no drug. This reduced cell death may indicate that the protamine also plays a biological role that improves neuron cell survival under normal in vitro culture conditions. Additionally, protamine significantly improved neuron adhesion to inhibitory substrates and showed viability as both a soluble and immobilized therapeutic. While astrocytes did appear to become more reactive in the presence of protamine, they did not favor a neuroinhibitory or neuroregenerative phenotype. Additionally, protamine did not influence CSPG production in astrocytes. Together, these findings show the toxicity threshold of PRM with neuronal and glial cells, as well as its preliminary in vitro therapeutic benefits. Future work should investigate biomaterial integration of protamine to facilitate controlled release or prevent fastidious clearance and systemic exposure of the drug. Data also shows that protamine may have a biological role in preventing innate neuron cell death during in vitro culture. This underlying mechanism will have to be elucidated in follow-up experiments and may further promote the application of protamine for neural tissue engineering challenges.

Acknowledgements (Optional): :

NIA Training Grant T32AG057464 to D.W.N., NSF GRFP DGE-1744655 to J.L.F., Rensselaer’s Graduate Fellowship to C.M., and NIH R01 NS092754 to R.J.G.

References (Optional): :

1. Jones, L.L.; Margolis, R.U.; Tuszynski, M.H. The Chondroitin Sulfate Proteoglycans Neurocan, Brevican, Phosphacan, and Versican Are Differentially Regulated Following Spinal Cord Injury. Experimental Neurology 2003, 182, 399–411, doi:10.1016/S0014-4886(03)00087-6.

2. Hyatt, A.J.T.; Wang, D.; Kwok, J.C.; Fawcett, J.W.; Martin, K.R. Controlled Release of Chondroitinase ABC from Fibrin Gel Reduces the Level of Inhibitory Glycosaminoglycan Chains in Lesioned Spinal Cord. Journal of Controlled Release 2010, 147, 24–29, doi:10.1016/j.jconrel.2010.06.026.

3. Loers, G.; Liao, Y.; Hu, C.; Xue, W.; Shen, H.; Zhao, W.; Schachner, M. Identification and Characterization of Synthetic Chondroitin-4-Sulfate Binding Peptides in Neuronal Functions. Sci Rep 2019, 9, 1064, doi:10.1038/s41598-018-37685-2.