Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons
Introduction
2,4-diaminobutyric acid (α,γ-diaminobutyric acid; 2,4-DABA) is a non-protein neurotoxic amino acid widely present in the environment. Originally identified as a metabolic product of bacteria and a component of polymixin antibiotics (Catch et al., 1948), 2,4-DABA has since been found both in animal samples, including mammalian brain (Nakajima et al., 1967), and in plants, some of which linked to neurotoxicity in humans such as several Lathyrus species (Ressler et al., 1961; Vanetten and Miller, 1963).
Recently 2,4-DABA was shown to be present in a wide variety of aquatic ecosystems: freshwater and marine cyanobacterial samples (Bishop et al., 2018; Faassen et al., 2009; Spacil et al., 2010), water plants (Al-Sammak et al., 2014), microalgae (Reveillon et al., 2016), marine microbial mats (Chatziefthimiou et al., 2018) plankton, peryphyton (Reveillon et al., 2015), bivalve organisms (Chatziefthimiou et al., 2018; Reveillon et al., 2016), fish from marine demersal, benthic and pelagic habitats (Chatziefthimiou et al., 2018), freshwater fish (Al-Sammak et al., 2014; Banack et al., 2015), crabs (Field et al., 2013) and lobsters (Banack et al., 2014). Many of aquatic species are capable of bioaccumulating the toxin (Jonasson et al., 2010; Reveillon et al., 2015).
Finally, 2,4-DABA is present in aerosol and terrestrial samples ranging from lakesides (Banack et al., 2015) to dust derived from air-dried desert crusts and mats (Cox et al., 2009; Metcalf et al., 2015) and desert soil (Chatziefthimiou et al., 2016).
Neurotoxic hallmarks of 2,4-DABA were recognized soon after its discovery. Riggs et al. (Riggs et al., 1954) were the first to observe preconvulsive behavior, convulsions and death in rats upon acute subcutaneous administration of the amino acid, and also focal degenerative changes of Purkinje cells of the cerebellum and ganglion cells of the rat cortex after chronic administration of 2,4-DABA. Following research confirmed these results and, more importantly, showed that oral administration to rats can lead to neurotoxic manifestations which involved hyperirritability, motor incoordination, paresis or paralysis of hind limbs (Vivanco et al., 1966), tremor of the forelimbs, convulsions and death (Ressler et al., 1961). The interest in neurotoxicity of DABA has recently been reinvigorated through recognition of its possible link to the Western Pacific amyotrophic lateral sclerosis – Parkinsonism/dementia complex (ALS/PDC).
ALS/PDC is a disorder characterized by unusually high prevalence of amyotrophic lateral sclerosis among the indigenous people of Guam, other Mariana Islands, and in the Kii Peninsula (Kurland and Mulder, 1954). In addition to ALS-like symptoms, patients also exhibit parkinsonian and dementia symptoms, hence the name amyotrophic lateral sclerosis – Parkinsonism/dementia complex. Initial research suggested that the cause of the symptoms resulted from ingestion of amino acid beta-N-methylamino-L-alanine (BMAA). The afflicted populations consumed BMAA of cyanobacterial origin, through a traditional diet rich in flour produced from seeds of the cycad plant (which forms symbiosis with cyanobacteria), and in much greater concentrations, presumably through biomagnification, from the flesh of animals that consume the seeds (Banack and Cox, 2003; Cox et al., 2003; Cox and Sacks, 2002). Whether Western Pacific ALS/PDC is truly caused by BMAA exposure remains controversial, but inquiry into BMAA distribution in cyanobacterial samples led to the discovery of its co-occurrence with 2,4-DABA, a structural isomer of BMAA, not only in cycad seeds (Pan et al., 1997) but also in most of the tested specimens worldwide (Faassen et al., 2009; Kruger et al., 2010; Rosen and Hellenas, 2008; Spacil et al., 2010). This opens the possibility of DABA involvement in neurodegenerative disorders in animals and men beyond the Western Pacific region. Global spreading of cyanobacteria (Svircev et al., 2019) and rising occurrence of harmful cyanobacterial blooms (Huisman et al., 2018) increase the risk of human exposure to cyanobacterial neurotoxins, including 2,4-DABA, making their impact more significant.
However, many aspects of DABA neurotoxicity remain unknown, including the incomplete understanding of mechanistic data on this amino acid. One of the first reports on mechanisms of 2,4-DABA induced neurotoxicity proposed that neuronal damage is not caused by DABA itself, but due to accumulation of liver-derived toxins in the brain (O’Neal et al. (1968)). It has since been shown that 2,4-DABA passes the blood-brain barrier and affects the neurons directly (Chen et al., 1972; Vivanco et al., 1966).
The most detailed account of possible mechanisms of 2,4-DABA toxicity comes from studies investigating potential use of 2,4-DABA as an anti-tumor agent. It has been shown that 2,4-DABA leads to irreversible damage in mouse fibrosarcoma (Ronquist et al., 1980), human and rat glioma (Panasci et al., 1988; Ronquist et al., 1984), human and mouse neuroblastoma (Main and Rodgers, 2018; Takser et al., 2016), and hepatoma (Blind et al., 2000, 2003) cell lines, murine blastocysts (Naeslund et al., 1979) and human fibroblasts (Panasci et al., 1988). Most of these in vitro studies suggest that the cytolytic effect of 2,4-DABA was caused by non-saturated transport of the amino acid into the cell by alanine-preferring sodium-dependent amino acid System A transporter (SAT), causing osmotic overload and cell death. In none of these studies electrophysiological investigations were performed.
To the best of our knowledge, the only study that reports mechanisms of 2,4-DABA induced electrophysiological effects was performed by Weiss et al., but only as a comparison to the effects of BMAA, which was the main topic of the paper. Intracellular recordings showed cell membrane depolarization, which was attenuated by kynurenate, suggesting mediation by ionotropic glutamate receptors (iGluRs) (Weiss et al., 1989a).
Considering ubiquitous presence of 2,4-DABA in the environment, proven neurotoxic properties, potential link to neurodegenerative disorders, and incomplete understanding of the mechanisms underlying its actions, especially electrophysiological, in this paper we have investigated possible mechanisms that may lead to previously reported (Spasic et al., 2018) damaging electrophysiological effects of 2,4-DABA on Retzius neurons of the leech Haemopis sanguisuga.
Section snippets
Materials and methods
Investigations were performed on Retzius neurons in isolated segmental ganglia of the leech ventral nerve cord, at room temperature (22−25 °C). The leeches Haemopis sanguisuga were acquired from a local distributor and maintained in aquaria at +4°, in dechlorinated tap water which was changed twice a week.
All procedures related to the use of animals were conducted in compliance with ethical guidelines recommended by institutions’ and national ethical boards. Methods of animal dissection have
Effect of DL-2,4-DABA on Retzius nerve cell membrane potential in sodium-free environment
As mentioned before, two mechanisms of direct 2,4-DABA action on neurons proposed in the literature are activation of ionotropic glutamate receptors (iGluRs) and activation of neutral amino acid transport system A (SAT). Since both of these mechanisms are sodium dependent, in the first group of experiments we have examined the changes of membrane potential of leech Retzius neurons exposed to DL-2,4-DABA in sodium-free (TRIS-Ringer) solution in order to inspect the sodium dependence of membrane
Discussion
2,4-diaminobutyric acid (2,4-DABA) is an excitatory neurotoxic amino acid present worldwide in virtually all environments, but predominantly in aquatic ecosystems. Recent studies suggest that 2,4-DABA may be a risk factor for development of neurodegenerative diseases in animals and humans. The mechanism by which this amino acid causes structural and functional damage of neurons is still unclear. Previous studies have shown that L-DABA may damage cells indirectly via ammonia accumulation
Conclusion
Taken together, all these results indicate that the electrophysiological effect of 2,4-DABA on Retzius neurons of the leech Haemopis sanguisuga is mediated by two distinct mechanisms – the first stage of depolarization by activation of ionotropic glutamate receptors, and the second stage by activation of a sodium-dependent electrogenic neutral amino acid transport system. Both of these could play a significant role in DABA induced neurotoxicity by triggering excitotoxicity, induction of osmotic
CRediT authorship contribution statement
Svetolik Spasic: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Marija Stanojevic: Conceptualization, Methodology. Jelena Nesovic Ostojic: Methodology, Writing - review & editing. Sanjin Kovacevic: Validation. Jasna Todorovic: Visualization. Marko Dincic: Visualization. Vladimir Nedeljkov: Conceptualization, Investigation, Resources. Milica Prostran: Funding acquisition. Srdjan Lopicic:
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was supported by The Ministry of Education, Science and Technological Development of the Republic of Serbia [grant number 175023] and COST action ES 1105.
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