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tion in neurological disorders. Assuming that the results with QYNAD

are confirmed, many important ques-tions remain. Do QYNAD concentrationsin the cerebrospinal fluid fluctuate and,if so, are they correlated with clinical sta-tus? To what degree does QYNAD (whichmay be one of several mediators of ax-onal dysfunction in MS and GBS) con-tribute to the overall clinical picture?Might immunization with QYNADinduce the production of antibodiesagainst QYNAD? And, if so, what effectswould there be on sodium channel func-tion and on neurologic status?

At least ten genes encode molecularlydistinct sodium channels. Eight (or more)of these are expressed within the nervoussystem. These different channel isotypeshave different pharmacological as well asphysiological profiles8. The sodium chan-nel isotypes that are expressed in myeli-nated and demyelinated axons in variousnerves and tracts are still being identified.The essential residues or ‘receptor’ forQYNAD within sodium channels have notbeen identified, and it is not yet clearwhich channel isotypes are blocked byQYNAD. A hint that QYNAD might have differential effects on the varioussodium channel isotypes comes from stud-ies that show a differential effect of lido-caine on different types of neuronalsodium channels9.

The presence of ‘endocaines’ may alsohave consequences for our understandingof the function of the normal nervous sys-tem. There is a growing body of evidenceindicating that neurons are dynamic elec-trogenic machines, capable of modulatingand/or remodeling their electrically ex-citable membranes (and their constituent

ion channels) so as to adjust their elec-troresponsive properties in response toshifting functional needs10. For example,sodium channels can be modulated byphosphorylation through cAMP-depen-dent protein kinase, protein kinase C andtransmembrane receptor tyrosine phos-phatase, which may also help to localizechannels within neuronal membranes11,12.Neurons may also respond to changes intheir input by differentially upregulatingand downregulating transcription of thegenes for various sodium channel isotypesso as to change the electrogenic propertiesof their membranes13. Physiological varia-tions in ‘endocaine’ levels within normalindividuals would add another mecha-nism for modulating neuronal excitability.

The description of QYNAD may not bethe end of this story. Other researchgroups are reportedly examining addi-tional aspects of ion channel functionthat may be perturbed in the demyelinat-ing diseases. New molecular mecha-nisms, in some cases involving channels,will probably soon be identified in MSand GBS and this, in turn, may presenttherapeutic opportunities. An importantlesson of the past decade has been that‘demyelinating’ diseases involve morethan damage to myelin. As part of thislesson we may soon learn that ion chan-nels, which are pivotal in normal neu-ronal function, are important targets inthese disorders.

1. Waxman, S.G. Demyelinating diseases: New patho-logical insights, new therapeutic targets. N. Engl. J.Med. 338, 323–325 (1998).

2. Brinkmeier, H., Aulkemeyer, P., Wollinsky, K.H. &Rodel, R. An endogenous pentapeptide acting as asodium channel blocker in inflammatory autoim-mune disorders of the CNS. Nature Med. 7, 808–811(2000).

3. Sakurai, M., Mannen, T., Kanazawa, I. & Tanabe, H.Lidocaine unmasks silent demyelinating lesions inmultiple sclerosis. Neurology 42, 2088–2093 (1992).

4. Takigawa, T. et al. Antibodies against GM1 ganglio-side affect K+ and Na+ currents in isolated rat myeli-nated nerve fibers. Ann. Neurol. 37, 436–442 (1995).

5. Vassilev, P.M., Scheuer, T. & Catterall, W.A.Identification of an intracellular peptide segment in-volved in sodium channel inactivation. Science 241,1658–1661 (1988).

6. Redford, E.J., Kapoor, R. & Smith, K.J. Nitric oxidedonors reversibly block axonal conduction: demyeli-nated axons are especially susceptible. Brain 120,2149–2157 (1997).

7. Renganathan, M., Cummins, T.R., Hormuzdiar,W.N., Black, J.A. & Waxman, S.G. Nitric oxide is anautocrine regulator of Na+ currents in axotomized C-type DRG neurons. J. Neurophysiol. 83, 2431–2443(2000).

8. Catterall, W.A. Cellular and molecular biology ofvoltage-gated sodium channels. Physiol. Rev. 72,S15–S48 (1992).

9. Cummins, T.R. & Waxman, S.G. Down-regulation oftetrodotoxin-resistant sodium currents and up-regu-lation of a rapidly repriming tetrodotoxin-sensitivesodium current in small spinal sensory neurons fol-lowing nerve injury. J. Neurosci. 17, 3503–3514(1997).

10. Waxman, S.G. The neuron as a dynamic electrogenicmachine: Modulation of sodium channel expressionas a basis for functional plasticity in neurons. Phil.Trans. R. Soc. Lond. B 355, 199–213 (2000).

11. Ratcliffe, C.F., Qu, Y., McCormick, K.A., Tibbs, V.C.,Dixon, J.E., Scheuer, T. & Catterall, W.A. A sodiumchannel signaling complex: modulation by associ-ated receptor protein tyrosine phosphate. NatureNeurosci. 3, 437–444 (2000).

12. Salter, M.W. & Wang, Y.T. Sodium channels developa tyrosine phosphatase complex. Nature Neurosci. 3,417–419 (2000).

13. Tanaka, M., Cummins, T.R., Ishikawa, K., Black,J.A., Ibata, Y. & Waxman, S.G. Molecular and func-tional remodeling of electrogenic membrane ofhypothalamic neurons in response to changes intheir input. Proc. Natl. Acad. Sci. USA 96,1088–1093 (1999).

1Department of Neurology and PVA/EPVANeuroscience Research CenterYale Medical SchoolNew Haven, Connecticut 06510, USA2Rehabilitation Research Center, VA HospitalWest Haven, Connecticut 06516, USAEmail: stephen.waxman@yale.edu

THE DEGENERATION AND death of neuronsin disorders such as Alzheimer dis-

ease, Parkinson disease, stroke and severeepileptic seizures is believed to result, inpart, from overactivation of receptors forthe excitatory neurotransmitter gluta-mate1,2. The neuronal loss in such disor-ders belies the fact that the nervoussystem has a remarkable ability to with-stand and adapt to injury. Research dur-ing the past 15 years has identified

several cellular signaling pathways thatare activated by injury and can promotesurvival and plasticity of neurons duringdevelopment as well as in the adult.Production of an array of neurotrophicfactors and cytokines increases consider-ably after traumatic, ischemic and othertypes of insults, and several of these fac-

tors have been shown to protect neuronsin experimental cell culture and animalmodels. For example, basic fibroblastgrowth factor (bFGF), brain-derived neu-rotrophic factor and tumor necrosis fac-tor-α protect neurons against damageinduced by severe seizures and cerebralischemia2. The neuroprotective factorsactivate cell surface receptors linked tokinase cascades that ultimately induceexpression of proteins such as antioxi-

Activin to the rescue for overexcited neuronsOveractivation of receptors for the excitatory neurotransmitter glutamate may be involved in disorders ranging fromepilepsy to Alzheimer disease. This destructive pathway may be held in check by basic fibroblast growth factor acting

through an intermediary cytokine called activin A (pages 812–815)

MARK P. MATTSON

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dant enzymes and proteins that stabilizecalcium homeostasis.

One prominent injury-responsivefamily of cytokines is named for its pro-totypical member, transforming growthfactor-β (TGF-β), which is produced byneurons and microglial cells. TGF-β hascomplex functions in neuronal and glialcell responses to injury but in generalseems to promote neuron survival3. Newfindings reported in this issue of NatureMedicine4 provide evidence that activinA, a member of the TGF-β family, has aneuroprotective function in a rodentmodel of epileptic seizure-induced braindamage. Activins and their inhibitorybinding partners (inhibins) have beenbest characterized in endocrine systems,in which they regulate hormonal feed-back systems5. Tretter et al. show that in-traventricular administration of theseizure-inducing excitotoxin kainate inmice results in increased levels of activinA mRNA in neurons in the CA1 region ofthe hippocampus, and that the increasein activin A expression is greatly en-hanced by co-administration of bFGF(ref. 4). Kainate-induced seizures causedegeneration of CA3 neurons, and suchdegeneration is prevented by administra-tion of bFGF. The involvement ofincreased activin A levels in the neuro-protective effects of bFGF is indicated bytwo findings: administration of exoge-nous activin A protects CA3 neurons,and administration of follistatin, a pro-tein known to inhibit activin A, abol-ishes the neuroprotective effect of bFGF.These findings are intriguing becausethey provide evidence for a previouslyunknown intermediary signal in the neu-

roprotective action of bFGF.Moreover, they identify activin Aand its signaling pathway as a tar-get for therapeutic intervention inexcitotoxic neurodegenerativeconditions.

Previous studies have shownthat bFGF can act directly on hip-pocampal neurons to protectthem from excitotoxic injury6. Itis therefore unclear why activin Aproduction is required for theneuroprotective effect of bFGF invivo. If activin A is required for theneuroprotective effect of bFGF inthe seizure model, then it may ormay not be required in the manyother models in which bFGF hasbeen shown to be neuroprotec-tive2. Although the findings re-

ported here indicate that activin Aprevents neuronal death4, previous stud-ies have shown that activin A can induceapoptosis of non-neuronal cells7. Thisscenario (protection of neurons andkilling of mitotic cells) is reminiscent offindings obtained in studies of tumornecrosis factor-α (ref. 3), another cy-tokine that has complex functions inneuronal injury responses3. Antagonisticeffects of activin A and bFGF on neuralcells have been documented8. Althoughthe data here4 are consistent with an im-portant function for production of ac-tivin A in the neuroprotective effect ofbFGF, there are additional questionsthat must be answered to properly inter-pret these findings.

There is discordance in the relation-ship between the neuronal populationsin which activin A is expressed and thepopulations that are protected by bFGF.Activin A expression is extremely low inCA3 neurons in basal conditions andafter kainate and bFGF administration,but activin A is present at very high lev-els in CA1 neurons. If activin A is essen-tial for the survival of CA1 neurons, thenfollistatin might therefore be expected torender CA1 neurons vulnerable tokainate-induced damage. Indeed, CA1neurons are sensitive to kainate-induceddamage, particularly in conditions inwhich neuroprotective mechanisms arecompromised9,10. One possible explana-tion for these data is that activin A pro-duced in CA1 neurons activates receptorslocalized in axons of the CA3 neuronsthat form synapses on CA1 neurons(Fig.1). Tretter et al. did not establish thecellular distribution of activin A recep-

tors in basal conditions or after adminis-tration of kainate or bFGF. It is thereforeunclear whether CA3 neurons are di-rectly responsive to activin A or whetherits action is indirect. Indeed, it is con-ceivable that activin A, as well as bFGFand follistatin, alter seizure activityrather than directly affecting the survivalof CA3 neurons.

The mechanism of action of activin Ais not yet known. Researchers will alsoneed to determine whether the ob-served antagonism of the neuroprotec-tive effect of bFGF by follistatin is due tospecific inhibition of activin A; follis-tatin might also endanger hippocampalneurons in conditions in which activinA is not involved, an important controlexperiment not done by Tretter et al. Amore-specific means of blocking activinA expression (antisense approaches orknockout mice) or action (receptorblocking antibodies) would help clarifythese issues.

The identification of this new anti-apoptotic signaling cascade initiated bybrain injury and bFGF provides a new‘window’ into the intricate mechanismswhereby the brain attempts to preserveits precious neurons. The stimulation ofactivin A production by bFGF provides abifurcating amplification system thatensures a high level of activation of cell-survival-promoting intercellular signal-ing. Because bFGF has proven effectivein reducing damage to brain cells andimproving functional outcome in exper-imental models of stroke and traumaticbrain and spinal cord injury, the newfindings on the neuroprotective func-tion of activin A indicate new possibili-ties for therapy. Administration ofactivin A is one obvious approach thatmerits further testing in preclinical stud-ies. Another approach would be to de-velop drugs that stimulate the signalingpathways activated by activin A. Timewill tell whether the potential of activinA indicated by the present findings is ul-timately fulfilled.

1. Beal, M.F. Aging, energy, and oxidative stress inneurodegenerative diseases. Ann. Neurol. 38,357–366 (1995).

2. Mattson, M.P. Neuroprotective signal transduc-tion: relevance to stroke. Neurosci. Biobehav. Rev.21, 193–206 (1996).

3. Mattson, M.P. et al. Cellular signaling roles ofTGFβ, TNFα and βAPP in brain injury responsesand Alzheimer’s disease. Brain Res. Rev. 23,47–61 (1997).

4. Tretter, Y.P. et al. Induction of activin A is essen-tial for the neuroprotective action of bFGF invivo. Nature Med. 6, 812–815 (2000).

5. Mather, J.P., Moore, A. & Li, R.H. Activins, inhib-

CA1ActA

bFGF

Seizuresand injury

AstrocyteCA3

ActA

Fig. 1 Severe seizures and related types of brain in-jury, including stroke, induce the production of basic fi-broblast growth factor (bFGF) by astrocytes andneurons. bFGF, in turn, stimulates production of activinA (ActA) in neurons. In the hippocampus, ActA pro-duced by CA1 neurons may activate receptors in CA3neurons and thereby protect them against injury. ActAmay also act directly on the neurons that produce it, inan autocrine manner.

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ESCHERICHIA COLI IS the prevalent etiologicalagent of pyelonephritis and other uri-

nary tract infections (UTIs) in humans.Most uropathogenic E. coli (UPEC) isolatessecrete α-hemolysin, a cytolytic toxin be-longing to the family repeats in toxin(RTX) found in a variety of Gram-negativebacterial pathogens1. This toxin con-tributes to the virulence of UPEC strains,as shown in several animal models, but itsprecise function in the pathogenesis ofUTIs is unclear. A paper by Uhlén et al. inthe 8 June issue of Nature2 reported that E.coli α-hemolysin is capable of inducing os-cillatory calcium signals in host cells thattrigger an inflammatory response that islikely to contribute to the clinical mani-festations of UTI.

E. coli α-hemolysin (HlyA) is synthe-sized as a protoxin of 110 kDa that ispost-translationally activated with theaid of the co-synthesized acyltransferaseHlyC by covalent acylation of two inter-nal lysine residues (Fig. 1). The activatedtoxin is secreted from E. coli by a specifictransport apparatus composed of threemembrane proteins (HlyB, HlyD andTolC). Probably outside the E. coli cell,calcium ions bind to a tandem array ofnonameric repeats present in the HlyAprotein. The calcium-binding domainthereby folds into a parallel β-roll struc-ture that is, in addition to the acylation,required for the interaction of α-hemolysin with the plasma membrane oftarget cells. Insertion of α-hemolysininto the cell membrane results in the for-mation of transient, cation-selectivetransmembrane pores, which may causecell death and osmotic cell lysis1.

In vitro, E. coli α-hemolysin showsstrong cytotoxic and cytolytic activityagainst a variety of human and animalcells. In addition, a range of reactions aretriggered in various cells when they are

exposed to sublytic doses of the toxin3–6.These α-hemolysin-induced cellular re-sponses include, for example, the releaseof inflammatory lipid mediators fromleukocytes and platelets, and the secre-tion of the pro-inflammatory cytokineinterleukin (IL)-1 from monocytes.Moreover, α-hemolysin induces an ox-idative burst and degranulation in poly-morphonuclear leukocytes and triggersseveral activities in endothelial cells.

Calcium is central as an intracellularsecond messenger and is involved in theregulation of a diverse array of cell func-tions7,8. To act as a signal, the concentra-tion of cytosolic calcium ions ([Ca2+]i)must increase into the micromolarrange. However, prolonged increases of[Ca2+]i can be lethal. Therefore, cells useeither sustained low-amplitude calciumsignals or, more commonly, deliver thecalcium signals as brief ‘transients’.These transients may appear as single

transients or as repetitive [Ca2+]i oscilla-tions8,9. Calcium oscillations increase theefficacy of signaling, and their frequen-cies contribute to the specificity of geneactivation10.

Uhlén et al. found low-frequency cal-cium oscillations in primary renal ep-ithelial cells that were exposed to anUPEC strain expressing α-hemolysin,and demonstrated that the secreted α-he-molysin is the inducer of this calcium re-sponse. The calcium oscillations had aconstant periodicity of 12.0 ± 0.7 min-utes, with [Ca2+]i increasing from a basallevel of about 0.1 µM to 0.5–1.5 µM. Theauthors further found that in a renal ep-ithelial cell line the production of thepro-inflammatory cytokines IL-6 and IL-8 was stimulated by the α-hemolysin-induced [Ca2+]i oscillations.

Free calcium ions in the cytoplasm canbe derived from sources outside the celland from stores within the endoplasmicreticulum. Calcium can enter cells bypassing through voltage-, receptor- orstore-operated calcium channels presentin the plasma membrane, and it can be re-

Dangerous signals from E. coli toxinα-hemolysin is a pore-forming cytolysin involved in the virulence of uropathogenic Escherichia coli strains. Recentstudies have shown that at sublytic concentrations, this toxin can also induce calcium oscillations that affect gene

expression, the inflammatory response and other cellular activities.

ALBRECHT LUDWIG1 & WERNER GOEBEL2

Fig. 1 Linear model of E. coli α-hemolysin (HlyA). The 110-kDa α-hemolysin protoxin is activated in thecytosol of E. coli by covalent, quantitative acylation of two lysine residues, Lys564 and Lys690. Thisprocess is directed by the co-synthesized protein HlyC. Secretion of the activated toxin is accomplishedby a specific ‘ABC exporter’ and depends on a transport signal located within the C-terminal 60 aminoacids of HlyA. A series of 13 tandem nonameric repeats with the consensus sequence LXGGXG(N/D)DXare located away from the acylation sites of HlyA. The binding of calcium to these repeats, which occursmost likely after secretion, induces the repeat domain to fold into a parallel β-roll. Both the acylation andthe calcium binding are required for HlyA to interact with target cell membranes and seem particularly tobe involved in membrane binding. Pore formation by E. coli α-hemolysin in target membranes dependson three hydrophobic domains and flanking sequences in the N-terminal half of the toxin.

N

Hydrophobic domainsTransport

signal

C

Fatty acids

Binding to target cell membranePore formation

Ca2+ - bindingrepeat domain

Lys564

Lys690

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ins, and follistatins: further thoughts on a grow-ing family of regulators. Proc. Soc. Exp. Biol. Med.215, 209–222 (1997).

6. Mattson, M.P., Murrain, M., Guthrie, P.B. &Kater, S.B. Fibroblast growth factor and gluta-mate: Opposing actions in the generation anddegeneration of hippocampal neuroarchitecture.J. Neurosci. 9, 3728–3740 (1989).

7. Chen, W., Woodruff, T.K. & Mayo, K.E. Activin A-induced HepG2 liver cell apoptosis: involvementof activin receptors and smad proteins.

Endocrinology 141, 1263–1272 (2000).8. Daadi, M., Arcellana-Panlilio, M.Y. & Weiss, S.

Activin co-operates with fibroblast growth factor2 to regulate tyrosine hydroxylase expression inthe basal forebrain ventricular zone progenitors.Neuroscience 86, 867–880 (1998).

9. Lason, W., Simpson, J.N. & McGinty, J.F. Mu butnot delta opioid receptor stimulation intensifieskainic acid-induced neurotoxicity in rat hip-pocampus. Neuropeptides 12, 89–94 (1988).

10. Bruce, A.J. et al. Altered neuronal and microglial

responses to brain injury in mice lacking TNF re-ceptors. Nature Med. 2, 788–794 (1996).

Laboratory of NeurosciencesNational Institute on Aging GerontologyResearch Center5600 Nathan Shock DriveBaltimore, Maryland 21224, USA

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