THE AUTOCRINE EXCITOTOXICITY OF ANTILLATOXIN, A NOVEL LIPOPEPTIDE DERIVED FROM THE PANTROPICAL MARINE CYANOBACTERIUM LYNGBYA majuscula by JOHN MICHAEL MOULTON (Under the D rect on of Dr. Thomas Murray) ABSTRACT Ant llatoxin (ATX) is a l popept de produce d by the mar ne cyanobacter um . Lyngbya majuscula . ATX, a Na channel act vator, produces N 0methyl 0D0aspartate (NMDA) receptor mediated neurotox c ty in rat cerebellar granule neurons (C2Ns). To determ ne whether ATX produced this neurotox c ty throu1h an ndirect mechanism, the nfluence of ATX on 1lutamate release 3as ascerta ned. ATX produced a concentrat on0 dependent increase in e,tracellular 1lutamate. This response was prevented by the Na . channel anta1onist tetrodotox n (TTX). ATX caused a stron1 membrane depolar 4at on 3 th a ma1nitude comparable to that of 100 mM KCL. ATX also produced concentrat on0dependent cytotox c ty as measured by lactate dehydro1enase act vity. Ca .2 influ, was measured us ng a fluorescent ima1 n1 plate reader (FLIPR). AT X produced concentrat on0dependent Ca .2 influx. The neurotox c mechanisms of ATX are therefore s m lar to those of brevetox ns, which produce neuronal injury through depolar 4at on0 nduced Na .1 load, glutamate release, rel ef of M1 .2 block of NMDA recepto rs, and Ca .2 influx. INDEX WORDS: Neurotox n, E,c toto, c ty, Glutamate, Sodium Channel, FLIPR, Cerebellar Granule Neurons THE AUTOCRINE EXCITOTOXICITY OF ANTILLATOXIN, A NOVEL LIPOPEPTIDE DERIVED FROM THE ANTROPICAL MARINE CYANOBACTERIUM LYNGBYA majuscula by JOHN MICHAEL MOULTON B.S., The Univers ty of 2eor1 a A Thes s Subm tted to the Graduate Faculty of The Univers ty of Geor1 a in Part al Fulf llment of the Requirements for the De1ree MASTER OF SCI ENCE ATHENS, GEOR2IA 2003 © 2003 John M chael Moulton All R 1hts Reserved THE AUTOCRINE EXCITOTOXICITY OF ANTILLATOXIN, A NOVEL LIPOPEPTIDE DERIVED FROM THE ANTROPICAL MARINE CYANOBACTERIUM LYNGBYA majuscula by JOHN MICHAEL MOULTON Ma9or Professor: Thomas Murray Comm ttee: 2aylen Edwards Jul e Coff eld Electronic Vers on Approved: Maureen 2rasso Dean of the 2raduate School The Univers ty of Geor1 a May 2003 v TABLE OF CONTENTS Pa1e LIST OF FI2URES@@@@@@@@@@@@@@@@@@@@@@@@@@@$/ CHAPTER 1 Introduct on........................................................................................................1 2 L terature Revie3 ...............................................................................................A 3 The Autocr ne E,c totox c ty Of Ant llatox n, A Novel L popept de Der ved From The Pantropical Mar ne Cyanobac ter um Lyngbya majuscula @@@$@@@@@@@@@@@@@@@@@@@@@@@8A REFERENCES ...............................................................................................................37 v LIST OF FIGURES Pa1e F 1ure 1: ATX 0Induced Cytotox c ty@@@@@@@@@@@@@@@@@@@@AC F 1ure 2: Membrane0Potent al Assay@@$$$@@@@@@@@@@@@@@@@@$AD F 1ure 3: V abil ty 0Assay@@@@@@@@@@@@@@@@@@@@@@@@$$$AB F 1ure 4: T me 0Dependent ATX 0St mulated Glutamate Efflux ........................................AE F 1ure 5: Concentrat on0Dependent ATX 0Mediated Glutamate Efflux ............................AF F 1ure 6: Correlat on Bet3een Glutamate Efflux and Cytotox c ty ..................................50 F 1ure 7: ATX 0St mulated Ca .2 Influx ..............................................................................51 F 1ure 8: Correlat on Bet3een Ca .2 Influx and Cytotoxic ty@@@@@@@@@@@C8 F 1ure 9: Pharmacolo1 cal Assessment of Glutamate Release@@@@@@@@@@$C> 1 CHAPTER 1 Introduction Mar ne cyanobacter a are ma9or producers of biolog cally act ve and structurally unique nat ural products (Or9ala, J. et al., 1995; Yokoka3a, F., et al., 1999; Berman, et al., 1999). These m croal1al blooms have been impl cated in human intox cat on and e,tens ve f sh mortal ty (Nogle et al., 2001). In addit on to the r ecolog cal s 1nif cance, m croalgal products possess great potent al as biochem cal and pharmacolog cal tools. The pantropical cyanobacter um, Lyngbya majuscula, produces a diverse array of secondary metabol tes, including, kal: tox n, debromoaphys atox n, lyn1byatox n, curac n 0A and ant llatox n, w th the latter be n1 the focus of this study. Ant llato, n is among the most ichthyotox c metabol tes isolated to date. In humans, symptoms of L. majuscula include, respiratory irr tat on, eye inflammat on, and severe contact dermat t s n e,posed f shermen and s3 mmers (Abal, 2001). A recent report has sho3n that ant llatox n nduces NMDA receptor0mediated neurotox c ty in pr mary cultures of rat cerebellar 1ranule cells (Berman et al., 1999). Th s was conf rmed morpholog cally as ATX 0e,posed C2Ns e,pressed s3ell n1 of neuronal somata, thinning of neur tes, and blebbing of neur te membranes. ATX 0 nduced neurotox c ty was concentrat on0 dependent w th an EC C0 of 20.1± 6.4 nM as monitored by lactate dehydrogenase efflux. Further char acter 4 ng ATXIs mechanism, L et al . (2001) showed that ATX acts as an act vator of volta1e 0gated Na . channels. They found that TTX, a Na . channel anta1onist, blocked the increase in ATX 0 nduced Na . nflux in C2Ns. They also used 2 J3HKBTX, a radiol 1and probe that labels receptor s te 2 on the volta1e 01ated Na . channel, to provide direct evidence for ATX interact on w th a s te on the α subunit of the Na . channel. Compet t ve binding assays revealed that ATX interacts w th e ther neurotox n s te 4 or an undiscovered novel s te. To determ ne directly 3hether ATX nduces a 1a n of funct on in the Na . channel, they used the 22 Na . flu, assay previously descr bed by Catterall and collaborators (Catterall, 1975; Tamkun and Catterall, 1981). They found that ATX el c ted a (TTX sens t ve) concentrat on0dependent st mulat on of 22 Na . influ, in C2Ns (ECC6M98.2 ± 12.0 nM). Our lab has recently sho3n that another class of mar ne al1al tox n, brevetox ns, produce acute e,c totox c ty in C2Ns throu1h an autocr ne mechanism. Brevetox ns are l pid 0soluble polyether neuroto , ns that produce per odic harmful algal blooms in the 2ulf of Me, co and west coast of Flor da (Baden, 1989). E,c toto, c ty is the e,cess ve st mulat on of neuronal glutamate receptors and resultant dysre1ulat on of cellular Ca .2 homeostas s that ult ma tely leads to cell death (Choi, 1988). Autocr ne e,c toto, c ty refers spec f cally to instances where e,c totoxic ty s a secondary consequence of 1lutamate efflux (Le st et al., 1997). Brevetox ns produce neuronal injury in C2Ns through depolar 4at on0evoked Na . load, 1lutamate efflux, rel ef of the M1 .2 block of the NMDA receptor, and subsequent Ca .2 influ,. Domoic ac d, a tr carboxyl c am no ac d analo1 of glutamate, is produced by the diatom Pseudo-nitzschia multiseries and produces a neurotox c resp onse in C2Ns that is mediated pr mar ly by NMDA receptors (Berman and Murray, 1997). Domoic ac d 0 nduced neurotox c ty was, however, demonstrated to be due to the r act vat on on AMPANka nate receptors. This act vat on results in membrane depolar 4at on, which 3 st mulates the release of 1lutamate and also rel eves the protect ve M1 .2 block in NMDA receptors. The present study was to test the hypothes s that ATX acts as an autocr ne e,c toto, cant by induc ng the release of 1lutamate in C2Ns. E,per ments 3ere carr ed out in a phys olog c medium at a temperature of 22°C. Reduced temperatures have previously been shown to increase the neurotox c potency of 1lutamate in cultured cerebellar 1ranule neurons (Berman and Murray, 1996). Moreover, pr mary culture d C2Ns are currently used as a model for study n1 both acute and delayed 1lutamate 0 nduced tox c ty (Schramm et al., 1990). This in vitro model provides a 90% homogeneous cell populat on e,press ng a 1lutamater1 c phenotype (Cox et al., 1990) and acquires some of the morpholog cal, biochem cal and electrophys olo1 cal character st cs of mature neurons (Jalonen et al., 1990). The present results conf rm that ATX tox c ty is due to the act vat on of volta1e dependent Na . channels. Moreover, 3e have sho3n that acute ATX cytotox c ty is assoc ated w th cellular s3ell ng, while delayed effects depend on the efflu, of endogenous 1lutamate. This was conf rmed morpholog cally by sta nin1 the neurons 3 th fluoresce n diacetate 0propidium iodide. Endogenous 1lutamate released into the media act vates both the NMDA and AMPANka nate receptors. As w th brevetox ns and domoic ac d, ATX may be vie3ed as capable of produc ng an autocr ne e,c toto, c ty in C2Ns. A CHAPTER 2 Literature Review Cyanobacteria and Their Toxins Cyanobacter a (blue0green algae) are amon1st the oldest spec es known to this planet. These photosynthet c, Gram 0ne1at ve, organisms are found both in mar ne and freshwater aquat c environments. Cyanobacter a typ cally 1ro3 best in warm, st ll, eu trophic or hypertrophic waters (Hunter 1998; Skulber1 et al. 1984). W th adequate m nerals, nutr ents, and a neutral or al:al ne pH, mass developments in aqueous suspens ons (blooms), surface scums and mats of cyanobacter a can be found. These blooms develop annually in the summer and autumn months. Though opt mal viabil ty is usually found in the temperate lat tudes, seasonal mats can also form in polar environments but arenIt as long l ved compared to the r temperate counterparts. Animal 0, bird 0, f sh0 and human 0poisonings have been ascr bed to cyanobacter al blooms and scums in the sc ent f c and popular press for over a century (Codd et al., 1994).