Nystatin Regulates Axonal Extension and Regeneration by Modifying the Levels of Nitric Oxide

An extremely simple question. WHOM IS GOING TO DO FOLLOWUP TO SEE IF THIS CAN GET ACROSS THE BLOOD BRAIN BARRIER?  NO ONE? YOU'RE SCREWED.  Deal with the incompetence of the stroke medical world, they have no clue what needs to be done until they are the 1 in 4 per WHO that has a stroke? Will that finally get you to do your job properly? Of course you will be disabled by then and lose your job so schadenfreude to you.

In vitro is used to describe work that's performed outside of a living organism. Or do we get around this problem by just increasing nitric oxide? WHOM WILL ANSWER THAT FUCKINGLY SIMPLE QUESTION?

Nystatin Regulates Axonal Extension and Regeneration by Modifying the Levels of Nitric Oxide

Cristina Roselló-Busquets^1,2, Marc Hernaiz-Llorens^1,2, Eduardo Soriano^1,2,3* and Ramon Martínez-Mármol^4*

• ¹Department of Cell Biology, Physiology and Immunology, Faculty of Biology and Institute of Neurosciences, University of Barcelona, Barcelona, Spain
• ²Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), ISCIII, Madrid, Spain
• ³Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
• ⁴Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute (QBI), University of Queensland, St Lucia Campus, Brisbane, QLD, Australia

Nystatin is a pharmacological agent commonly used for the treatment of oral, mucosal and cutaneous fungal infections. Nystatin has also been extensively applied to study the cellular function of cholesterol-enriched structures because of its ability to bind and extract cholesterol from mammalian membranes. In neurons, cholesterol level is tightly regulated, being essential for synapse and dendrite formation, and axonal guidance. However, the action of Nystatin on axon regeneration has been poorly evaluated. Here, we examine the effect of Nystatin on primary cultures of hippocampal neurons, showing how acute dose (minutes) of Nystatin increases the area of growth cones, and chronic treatment (days) enhances axon length, axon branching, and axon regeneration post-axotomy. We describe two alternative signaling pathways responsible for the observed effects and activated at different concentrations of Nystatin. At elevated concentrations, Nystatin promotes growth cone expansion through phosphorylation of Akt; whereas, at low concentrations, Nystatin enhances axon length and regrowth by increasing nitric oxide levels. Together, our findings indicate new signaling pathways of Nystatin and propose this compound as a novel regulator of axon regeneration.

Introduction

Mammalian adult Central Nervous System (CNS) differs from embryonic CNS and Peripheral Nervous System (PNS) by their inherent ability to regenerate lesioned tissues. After axotomy, the first regeneration step requires the formation of a functional growth cone. Unfortunately, the adult CNS has a reduced capacity to form new growth cones due, to the existence of intrinsic factors (Ertürk et al., 2007) and the presence of growth-inhibitory molecules (Tan et al., 2005; Li et al., 2013). After axotomy, organized sequential steps are required to form new and functional growth cones. The first of which consists of the influx of calcium, which increases exocytosis to fuse additional membrane to form a sealing patch to repair the ablated axon (Bradke et al., 2012; Blanquie and Bradke, 2018; Curcio and Bradke, 2018). Following this initial membrane addition, microtubule and actin cytoskeleton is reorganized, multiple signaling cascades are activated and the new membrane is transported to the tip of the growing axon (Bradke et al., 2012; He and Jin, 2016; Curcio and Bradke, 2018). A tight control of the actin cytoskeleton is crucial for the formation and functionality of the new growth cone. Regulation of actin requires the initiation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling cascade (Henle et al., 2011; Kakumoto and Nakata, 2013; Berry et al., 2016; Curcio and Bradke, 2018; Jin et al., 2018). Akt phosphorylation induces the activation of nitric oxide synthase (NOS), whose function is associated with actin reorganization and cell survival (Michell et al., 1999; Van Wagenen and Rehder, 2001; Welshhans and Rehder, 2005; Cooke et al., 2013; Sild et al., 2016). NOS produces nitric oxide (NO), a gaseous molecule involved in neurotransmission, neuronal growth and filopodia formation (Van Wagenen and Rehder, 2001; Welshhans and Rehder, 2005; Tojima et al., 2009; Forstermann and Sessa, 2012). NO is also associated with axon regeneration in insect neurons (Stern and Bicker, 2008) and the snail Helisoma trivolvis (Cooke et al., 2013). NO cannot be stored in cells, so its effects depend on the de novo synthesis by NOS activity. From the three types of NOS, neural NOS (nNOS) is synthesized in CNS and PNS neurons and its activity is regulated by intracellular calcium levels. The NO downstream signaling pathway involves the activation of protein kinase G (PKG) and actin-associated proteins such as the Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), resulting in a strong reorganization of the actin cytoskeleton (Zhou and Zhu, 2009; Forstermann and Sessa, 2012; Cossenza et al., 2014).

Nystatin is a drug commonly used as an antifungal agent because of its ability to destabilize fungal membranes by binding and extracting ergosterol, causing changes in cell permeability and, eventually, cell lysis (Bolard, 1986; Coutinho et al., 2004). Nystatin can also bind to cholesterol and extract this lipid from the membranes of mammalian cells. As a consequence, Nystatin has been widely used to disrupt and study the cellular function of lipid rafts. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, that facilitate the compartmentalization of signaling proteins, working as platforms for spatial and temporal regulation of the cytoskeleton, membrane anchoring, and cell adhesion, controlling the motility of growth cones (Guirland and Zheng, 2007), and the regenerative properties of lesioned axons (Tassew et al., 2014; Roselló-Busquets et al., 2019). The extended clinical use of Nystatin, together with its ability to affect the organization of lipid rafts, makes it an ideal candidate to explore its function as a possible therapeutic agent for the treatment of spinal cord lesions.

Here, we performed an in vitro evaluation of the Nystatin induced axonal regenerative properties, analyzing the effect of various concentrations and incubation times of this compound in hippocampal neurons. The study of the downstream signaling proteins responsible for the observed effects of Nystatin suggested that Nystatin differentially activates Akt phosphorylation and NO production in a concentration-dependent manner. We propose Nystatin as a novel neuronal pharmacological regulator of Akt and nNOS activity that modifies growth cone dynamics and promotes axonal regeneration post-axotomy.

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