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Na+ currents that fail to inactivate
reviews
Na ÷ currents that fail to inactivate Charles P. Taylor non-inactivating Na + current (also called sustained or CharlesP. Taylorisat persistent Na + current) has been characterized by theDeptof sensitivity to TTX in a variety of neurons, including Neuroscience squid axon 1, cerebellar Purkinje cells2 (Fig. 1), Pharmacology,Parkeneocortical pyramidal cells (Fig. 2) 3-5, thalamic DavisPharmaceutical Research,Divisionof neurons 6, CA1 hippocampal pyramidal cells7'8, striatal Wamer-LambertCo., neurons 8 and mammalian CNS axons9. Sustained Na + 2800PlymouthRd, currents have also been observed in glial cells 1° and in AnnArbor, several other excitable cells (see the discussion in ~HI48105,USA. Ref. 11). Most sustained Na + currents are blocked by TTX, by internal application of the permanently charged local anesthetic QX-314 or by application of solutions with reduced Na + concentration, and they are mostly insensitive to Ca 2+ channel blockers such as Ba2+, Co 2+, Mn z+ or Cd 2+. Sustained Na + current is typically a very small fraction of the peak inward current, and it has often been ignored in formal analyses such as the original study of Hodgkin and Huxley1~. In addition to the sustained current that is the subject of most of this review, there is a sustained Na + current predicted by the Hodgkin-Huxley analysis that occurs because the voltage dependence of Na + current activation and inactivation overlap. In contrast to non-inactivating current, this 'window current' only occurs over a narrow range of membrane voltages between about -50 and -40mV. In squid axon I and in adult mammalian pyramidal cells 3'4'7 non-inactivating Na + current represents 1-3% of the
Textbook accounts give the impression that Na + channels are short-acting binary switches: depolarization opens them, but only for about one millisecond. In contrast to this simplified view, a small but significant fraction of the total Na ÷ current in neurons occurs because channels open after long delays or in longduration bursts of openings. Such non-inactivating Na + current acts physiologically in neurons to amplify synaptic potentials and enhance endogenous rhythmicity, and also to aid repetitive firing of action potentials. In glial cells it also may regulate Na+-K + ATPase activity. The evidence for non-inactivating Na + current in a variety of neurons and glia is reviewed, along with a brief discussion of its ion channel substrate and its relevance for neurological diseases and drug therapy. Voltage-sensitive Na + channels are one of the basic building blocks of the nervous system, since they allow action potentials to propagate long distances down axons by causing regenerative and transient increases in Na + ion permeability. Several different classes of voltage-sensitive Na + channels have been identified, and some of these differ in their kinetics such as the rate of activation or inactivation or their sensitivity to the specific blocker tetrodotoxin (TTX). In contrast to differences in kinetics of various Na ÷ currents, a small fraction of Na ÷ current fails to inactivate even with prolonged depolarization. Such
A
B Mg2+, TTX
20 mV 2 nA i
I __
|
L-. •
,
-- L
:
I~
100 ms
Fig. 1. (A) Na+-dependent plateau potentials recorded by sharp electrode current-clamp from a Purkinje neuron in guinea-pig2+ cerebellar, tissue slice.2 Tissue was bathed with solution containing " /Hg2 + (3.7m/v1) and no Ca2 + to block Ca currents. Supenmposed traces show the results of transmembrane current pulses (150ms duration, ~ O. 17 nA, 0.31 hA, 0.42 nA, 0.82 nA and 2.0 nA) from the recording electrode. Rapid Na +-dependent action potentials are seen, but some traces also show plateau potentials of about 20 mV that outlast the applied current by up to 350ms. (B) Rapid action potentials and plateau potentials are abolished by application of tetrodotoxin (TTX) (301~). Note that the time constant to charge the membrane passively is approximately 30 ms, which is a much shorter period of time than the duration of plateau potentials. These findings indicate that plateau potentials are caused by a non-inactivating current from voltage-sensitive Na + channels. TINS, Vol. 16, NO. 11, 1993
© 1993, Elsevier SciencePublishersLtd, (UK)
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A
:
B
:
C
b
TTX
c
Fig. 2. Persistent Na + current was recorded from pyramidal tract neurons using a sharp intracellular microelectrode in slices of cat neocortex maintained in vitro 3. (A) Current-clamp records are compared with (B) voltage-clamp response to a slowly rising voltage ramp in the same neuron. In both parts, voltage is shown by the upper traces and current by the lower traces. Both (,4) and (B) show evidence of sustained Na + current. In (,4) transmembrane voltage responses are shown in response to three different amounts of applied current (trace 1, -O.33 nA; trace 2, +O.33 nA and trace 3, +O.67nA, each lasting for 200ms). Note that action potentials in trace 3 are preceded by a slower depolarizing potential (between lines b and a) that traverses the voltage range corresponding with a region of negative slope in the voltage-clamp record [inward rectification in (B)]. Line d indicates the resting membrane voltage (-75mY). (C) The negative slope region of voltage-clamp records is made finear by appfication of 11ZMtetrodotoxin (TTX), indicating that it is caused by voltage-sensitive Na + channels. Other experiments (not shown) demonstrate that the negative slope region is not altered by 2 mM COCI2, but is blocked by intracellular appfication of the local anesthetic QX-314.
peak amplitude of Na+current. Sustained current has sometimes been designated lNa p or INa (slow)because it persists for long periods of time, in contrast to the widely studied transient current. These designations imply that sustained current is due to activation of a separate class of Na + channels than those responsible for transient current, but recent single-channel studies (see below) argue against that view. The regenerative nature of sustained Na + currents explains long-lasting plateau potentials 2'4'8'13 (Fig. 1) and sub-threshold depolarizations 14 that are particularly prominent if repolarizing K + currents are blocked. Such Na+-dependent plateau potentials can last for seconds, much longer than the passive membrane-charging time constant. Thus, plateau potentials are caused by an inward current that fails to inactivate even with very long durations of depolarization. Sustained Na + current is responsible for inward rectification that causes a negative slope region in steady-state I-V curves in electrophysiological studies of neocortical pyramidal cells 3'4 (Fig. 2B) and hippocampal neurons 15. Such inward rectification is reduced by TTX or by intracellular application of the local anesthetic QX-314. As with rapidly inactivating Na ÷ current, sustained current is
A
regenerative: as channels become activated, the membrane depolarizes and additional channels are recruited. Recent studies 8 have revealed an extremely slowly activating and de-activating component of sustained inward current in isolated stfiatal and hippocampal neurons that is TTX-insensitive (Fig. 3). However, in agreement with studies of rapidly inactivating Na + current, the slow current is blocked by replacement of Na + ions with Tris ions and it is partly supported when Na + ions are replaced by K + ions. Nevertheless, the insensitivity of this current to "FI'X indicates that it is different from conventional Na ÷ currents, and this may be due to the action of a different type of Na + channel protein.
Ion channels responsible for sustained Na + current Several previous studies did not determine whether sustained Na + current is caused by a novel type of Na + channel or by the same type of channel responsible for transient Na + current 4,7. Recently, the single-channel analysis by Alzheimer et al. n has shown that membrane depolarization can cause three types of Na + channel gating in a single small
B 90 mM Na ÷
C
0 mM Na +, 0 mM K +, 90 mM Tris
90 mM K +
4oopr 90 ms
Fig. 3. Voltage-clamp records from isolated striatal neurons in vitro 8 show (A) a slowly activating inward current that is (B) blocked by replacement of Na + by Tris ions and is (C) partly supported in medium with 90mM K +. Experiments were performed in the presence of 40 mM tetraethylammonium, 4 mM 4-aminopyridine, 3 m M Mn 2+, no added Ca2+ and 12 ~ tetrodotoxin (TTX) to block K +, Ca2+ and rapid Na + currents. Insensitivity to TTX suggests that these sustained and kinetically very slow Na + currents are caused by a different kind of Na + channel than those causing currents shown in Figs 1 and2. Note that the peak ampfitude of these currents (approximately 3OOpA) is much less than those of rapidly inactivating Na + currents. 456
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2.0 m V membrane patch of acutely isolated neocortical neurons (Fig. 4). In addition to familiar short-latency RMP -'~----_.j ........................ ". . . . . . . . . . . . . . . . . . . . . . . . . transient openings following a depolarizing step, Na + - 5 0 m V channels can display infrequent brief openings that occur at relatively long delays after the start of depolarization. These 'delayed, brief openings' have durations similar to those of 'normal, rapid' Na ÷ current. The delayed brief openings probably account for a significant fraction of sustained current observed in whole-cell recordings. In the same patch of membrane that displays mostly transient Na + channel openings, about 1% of traces show very prolonged bursts of openings lasting tens of milliseconds or more (Fig. 4). These sustained bursts sometimes appear in 'blocks' or 'runs' that last for seconds. Similar prolonged bursts appear more commonly in cloned rat brain type-III Na ÷ channels that are physiologically expressed mainly in the immature brain TM. Prolonged bursts in both preparations have identical single-channel conductances and similar voltage dependence for activation to those observed for brief openings. In patches from acutely isolated brain cells u, the mean open time for Na ÷ channels during a prolonged burst was found to be voltage-dependent (0.5 mS at -60mV and 30mS at -20mV). The mean open time for delayed brief openings was also voltage dependent, but only within a much narrower range of durations. Because sustained bursts have such long durations, and because individual openings are longer with large depolarizations, prolonged bursts may contribute significantly to sustained Na* current recorded from whole cells, despite their relatively infrequent occurrence. The simplest interpretation of the single-channel data is that an individual Na ÷ channel can open either i~ :~ "': "i %~--~- :-'; :r'~¢i ~ - c - : ~'~- ::r ":':::" :--"::-': ",; j- ~'~'" immediately after depolarization (short-latency brief openings) or with a lower probability after a delay (delayed brief openings), but the same channel occasionally enters an open state that lacks fast inactivation. An alternative explanation for sustained single-channel gating is that a different class of Na ÷ channel proteins or a sub-population of altered channels co-exist with 'normal' channels in patches. The latter hypothesis supposes that the altered channels are usually silent (and thus are not detectable), but that they occasionally open in a sustained manner in the same patch of membrane that contains mostly normal channels. This possibility seems unlikely because sustained Na ÷ channel openings have been observed in a variety of other tissues besides the brain, including skeletal and cardiac muscle (see discussion in Ref. 11). They have also been observed in mammalian cells transfected with cDNA for rat brain type-IIA Na + channels ~7 and in X e n @ u s oocytes injected only with rat brain type-III Na ÷ channel mRNA 16. On statistical grounds Moorman et al. 16 have rejected the hypothesis that there is a different population of channels for sustained openings (a single Fig. 4. Voltage-clamp recordings from a small celltype of Na + channel might be altered even if attached patch on an acutely isolated rat neocortical expressed from a single mRNA by, for example, post- neuron ~1 show that most depolarizations cause rapid, translational processing). The authors of both recent brief channel openings. Occasionally, delayed brief opensingle-channel studies 11 ' 1(5" favor the idea that sus- ings (fourth trace down) or delayed but very prolonged bursts of openings (third trace from bottom) occur. All of tained openings occur interspersed with short- these types of channel openings are sensitive to tetroduration openings because of 'modal' gating, meaning dotoxin (not shown). Prolonged bursts and delayed brief occasional transitions of Na ÷ channels to a confor- openings may both contribute to the sustained currents mational state from which rapid inactivation is un- seen in whole-cell recordings such as those of Figs 1 and likely. 2. Abbreviation: RMP, resting membrane potential
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(1)
investigated. In cardiac cells, application of lysophosphatidylcholine (a metabolite of phospholipid breakdown) greatly enhances the fraction of sustained Na + current 2°, although the mechanism of this effect has not been established. Additional experiments will no doubt improve our knowledge of the mechanisms that modulate Na + channel gating to enhance or reduce sustained current.
Na +
[Na+ ]i [K+ ]i
-
K+ or
+
P h y s i o l o g i c a l f u n c t i o n of n e u r o n a l n o n - i n a c t i v a t i n g Na + c u r r e n t s
EPSPs, or responses to iontophoretic glutamate application in hippocampal pyramidal cells, increase in size when they occur within the voltage range of sustained Na + currents 2z'22. Thus, relatively slow synaptic potentials that otherwise would not elicit action potentials become effective. The amplification of sub-threshold depolarizations is greatly reduced by K+ TTX or by the intracellular administration of QX-314 Fig. 5. Model for Na + channel modulation of Na+-K + (Refs 21, 22). In photoreceptor cells and certain other ATPase activity in glial cells2e. Na + channels maintain neurons without prominent action potentials, nonintracellular [Na +] at levels that provide a substrate for Na +-K + exchange. Tetrodotoxin blocks Na + channels (1) inactivating Na + currents amplify small changes in and causes intracellular [Na +] to decrease to very low membrane potential 23. levels, inactivating Na+-K + exchange. In contrast, In the presence of sustained Na + currents, repetistrophanthidin and ouabain directly inhibit the A TPase tive action potentials are more likely and threshold protein (2). for regenerative action is set primarily by sustained Na + current 3'6'22. Thus, the sustained 'sub-threshold' Several studies 4'5'7'8'11 suggest that sustained Na + current may be a major determinant of the rate of currents occur on or close to pyramidal cell bodies. repetitive firing of action potentials. It could also be Thus, non-inactivating Na + current might be es- speculated that sustained Na + currents assist passive pecially prominent in type-I Na + channels that are current flow from one part of neuronal membrane to localized primarily on somata and near the primary another and thus spatially integrate synaptic potenbranches of large dendrites TM. However, voltage- tials, as has been proposed for sustained dendritic clamp experiments with cloned type-I Na + channel Ca2+ currents 24. proteins show rapid inactivation that is at least grossly Neocortical pyramidal cells display endogenous similar to that of type-IIA channels (Scheuer, T. and depolarizations that cause rhythmic firing of action Catterall, W. et al., pers. commun.). Non-inactivating potentials and that are sensitive to TTX 5,25. Thus, Na + currents are also present in peripheral nerves, non-inactivating Na + channels may cause endogenous where type-I Na + channels are rare 9. rhythmicity at about 7Hz, near the frequency of Most of the analyses discussed above are consist- 'theta' rhythm in EEG recordings. Sustained Na + ent with the idea that sustained Na + currents arise currents are one of a fairly large family of voltagefrom infrequent sustained gating of completely activated ionic currents that contribute to oscillations 'normal' Na + channels. However, the recent descrip- in membrane potential independent of action potention s of a slow TTX-insensitive Na + current in tials24. neurons of the mammalian brain indicates that at least a fraction of sustained current in some neurons is from N o n - i n a c t i v a t i n g Na + c u r r e n t s in glial cells a distinct population of ion channel proteins (see Recent work by Sontheimer and co-workers 26 above). suggests that non-inactivating Na + channels play an important role in gila, where action potentials do not M o d u l a t i o n of s u s t a i n e d Na + c u r r e n t s occur physiologically (and therefore rapidly inactiSustained Na + currents might be modified or vating currents are mostly irrelevant). Glia possess modulated by phosphorylation of channel proteins, voltage-dependent Na + channels, including a novel post-translational processing of channel proteins or by type that is similar in many respects to those in other intracellular messengers. Phosphorylation of neurons 27, although the total membrane conductance Na + channels by protein kinase C slows the time- is dominated by K + channels. One class of glial Na + course of Na + channel inactivation and makes sus- channel proteins is significantly less sensitive to TTX tained bursts of openings more common 17, but it has than neuronal channels. Electrophysiological studies not been established whether the fraction of non- show that sustained Na + currents are present in glial inactivating current in whole-cell recordings is in- cells 1°'2a, and sustained currents are the only ones creased by this treatment. Proteases such as trypsin expected under physiological conditions. or papain practically abolish rapid inactivation of Na + Treatment of glia in cell cultures with TTX causes channels when applied by an intracellular micro- slow membrane depolarization and prevents Rb + pipette, leaving an unusually large fraction of non- influx via the Na+-K + ATPase 26 (see Fig. 5). These inactivating Na + current 19. Endogenous cytoplasmic effects of TTX mimic but do not add to those of proteases might also cleave the inactivation gate of ouabain or strophanthidin (specific ATPase inhibitors). Na + channel proteins and thereby increase sustained It is proposed that the effects of TTX in glia stem Na + current, but this hypothesis has not been from loss of electrogenic Na+-K + pumping. The 458
TINS, VoL 16, No. 11, 1993
inhibition of ATPase activity by TTX is interpreted to mean that an inward 'leak' of Na + through voltagesensitive channels is necessary to allow the N a + - K + ATPase to exchange Na ÷ ions for external K + ions. When Na + influx is blocked with TTX, action of the Na+-K + pump lowers intracellular [Na +] so much that further exchange of the two ions can no longer occur, and the transmembrane K + gradient eventually decays. Sustained TTX application kills glial cells, presumably by inactivating the ATPase. Therefore, non-inactivating Na + channels in glia may provide the correct intracellular [Na+] for gila to perform their K+buffering function in brain tissues. It is further speculated26 that activation of Na + channels may increase glial Na+-K + ATPase activity by raising cytoplasmic [Na +] in response to glial depolarization. Anticonvulsants
The drugs phenytoin and carbamazepine interact with Na + channels in much the same way as lidocaine, by reducing the probability that Na + channels can open from normal resting potentials ~9'3°. When fast inactivation is removed by treatment with proteolytic enzymes, phenytoin still reduces the probability of channel opening at low micromolar concentrations 31, and so phenytoin and similar drugs are expected to inhibit sustained Na + currents. Results with cardiac myocytes32 also indicate that lidocaine mimics or stabilizes the 'slow inactivated' state of Na + channels. It will be interesting to determine whether anticonvulsants and local anesthetics inhibit sustained neuronal Na + currents at lower concentrations than they inhibit rapid Na + currents, as already demonstrated with local anesthetics in cardiac myocytes33 and with other cardiac Na + channel modulators such as the experimental compound R 56865 (Ref. 34) or with intraceUular application of QX-314 to neurons 4. Relevance
to ischemic
stroke
Several drugs known to modulate Na + channels prevent neuronal damage in models of brain ischemia. Although convincing evidence is still lacking, a variety of data suggest that sustained Na + currents might be involved. Neuroprotective Na+-modulating drugs include lidocaine 35-a7 and phenytoin36'38'39, as well as newer agents such as RS-874764° and BW-1003C8741. These compounds undoubtedly act at multiple sites, but each of them potently modulates voltagedependent Na + channels. In contrast to these drugs, TTX is very selective for Na + channels and it also prevents neuronal d a m a g e 36'37'42-45 and glutamate release 46 from hypoxia or ischemia in brain tissues. Neuroprotection with Na+-modulating drugs at concentrations that do not block action potentials 36'37 implies that sustained Na + currents may be involved, and sustained currents are known to be present in optic nerve 9 and in other CNS neurons'3 5 7' 8' where neuroprotection has been demonstrated. Other data also suggest that sustained Na + currents contribute to ischemic damage. Long bursts of action potentials that might contribute significant Na + influx during ischemia have not been described, but sustained depolarizations that would particularly favor non-inactivating Na + currents are often seen 47-49 . A m a s s i v e T T X - s e n s i t i v e Na + accumulation occurs in brain slices deprived of o x y g e n in vitro, and this Na + influx continues e v e n after action potentials c e a s e 5°. TINS, Vol. 16, No. 11, 1993
Such a large TTX-sensitive influx of Na + ions would require very large numbers of action potentials, but even a relatively small Na + current sustained over minutes could contribute a relatively large flux of Na + ions. A similar hypothesis for the role of noninactivating Na + current in ischemic damage has been advanced for cardiac myocytes5z, where a large fraction of toxic Caz+ entry appears to follow from increased cytosolic Na + concentration.
Selected references 1 Gilly, W. F. and Armstrong, C. M. (1984) Nature 309, 448-450 2 Uin&s, R. and Sugimori, M. (1980) J. Physiol. 305, 171-195 3 Stafstrom, C. E., Schwindt, P. C. and Crill, W. E. (1982) Brain Res. 236, 221-226 4 Stafstrom, C. E., Schwindt, P. C., Chubb, M. C. and Crill, W. E. (1985) J. NeurophysioL 53,153-170 5 Alonso, A. and Llin,~s, R. (1989) Nature 342, 175-177 6 Jahnsen, H. and Llin&s, R. (1984) J. Physiol. 349, 227-247 7 French, C. R., Sah, P., Buckett, K. J. and Gage, P. W. (1990) J. Gen. Physiol. 95, 1139-1157 8 Hoehn, K., Watson, T. W. J. and MacVicar, B. A. (1993) Neuron 10, 543-552 9 Stys, P. K., Sontheimer, H., Ransom, B. R. and Waxman, S. G. Proc. Natl Acad. 5ci. USA (in press) 10 Barres,B. A., Koroshetz, W. J., Chun, L. L. Y. and Corey, D. P. (1990) Neuron 5, 527--544 11 Alzheimer, C., Schwindt, P. C. and Crill, W, E. (1993) J. Neurosci. 13,660-673 12 Hodgkin, A. L. and Huxley, A. F. (1952) J. PhysioL 117, 500-544 13 Jahnsen, H. (1986) J. Physiol. 372, 129-147 14 MacVicar, B. A. (1985) Brain Res. 333,378-381 15 Hotson, J. R., Prince, D. A. and Schwartzkroin, P. A. (1979) J. NeurophysioL 42,889-895 16 Moorman, J. R., Kirsch, G. E., VanDongen, A. M. J., Joho, R. H. and Brown, A. M. (1990) Neuron 4, 243-252 17 Numann, R., Catterall, W. A. and Scheuer,T. (1991) Science 254, 115-118 18 Westenbroek, R. E., Merrick, D. K. and Catterall, W. A. (1989) Neuron 3,695-704 19 Gonoi, T. and Hille, B. (1987) J. Gen. Physiol. 89, 253-274 20 Undrovinas, A. I., Fleidervish, I. A. and Makielski, J. C. (1992) Circ. Res. 71, 1231-1241 21 Deisz, R. A., Fortin, G. and Zieglg~nsberger, W. (1991) J. Neurophysiol. 65, 371-382 22 Hu, G-Y. and Hvalby, O. (1992) Exp. Brain Res. 88, 485-494 23 Vallet, A, M., Coles, 3. A., Eilbeck, J. C. and Scott, A. C. (1992) J. Physiol. 456, 303-324 24 Llin,hs,R. (1988) Science 242, 1654--1664 25 Silva, L, R., Amitai, Y. and Connors, B. W. (1991) Science 251,432-435 26 Sontheimer, H., Fernandez-Marques, E., UIIrich, N. and Waxman, S. G. (1993) 5oc. Neurosci. Abstr. 19, 688 27 Gautron, S. et aL (1992) Proc. Natl Acad. Sci. USA 89, 7272-7276 28 Howe, J. R. and Ritchie, J. M. (1992) J. PhysioL 455,529-566 29 Catterall, W. A. (1987) Trends PharmacoL Sci. 8, 57-65 30 Ragsdale, D. S., Scheuer, T. and Catterall, W. A. (1991) 44ol. Pharmacol. 40, 756--765 31 Quandt, F. N. (1988) 44ol. PharmacoL 34, 557-565 32 Zilberter, Y., Motin, L., Sokolova, S., Papin, A. and Khodorov, B. (1991) J. 44ol. Cell CardioL 23 (Suppl. 1), 61-72 33 Ju, Y-K., Saint, D. A. and Gage, P. W. (1992) Br. J. PharmacoL 107, 311-316 34 Verdonck, F., Bielen, F. V. and Ver Donck, L. (1991) Eur. J. Pharmacol. 203, 371-378 35 Rasool, N., Faroqui, M. and Rubinstein, E. H. (1990) Stroke 21,929-935 36 Taylor, C. P. and Weber, M. (1992) Soc. Neurosci. Abstr. 18, 1135 37 Stys, P. K., Ransom, B. R. and Waxman, S. G. (1992) J. Neurophysiol. 67, 236--240 38 Clifton, G. L, Taft, W. C., Blair, R. E., Choi, S. C. and DeLorenzo, R. J. (1989) Stroke 20, 1545-1552 39 Boxer, P. A. et al. (1990) Stroke 21 (Suppl. III), 47-51 40 Alps, B. J. (1992) Br. J. Clin. Pharmacol. 34, 199-206
Acknow/edgernents I wishto thank BilICatterall, BrianMacVicar, HaraldSonb~eimer and PeterStys for providing unpublished manuscriptsand informab'on.Special thanks to David Rock andJeffery Kocsisfor helpful discussions and criticismof the manuscript. 459
41 Meldrum, B. S. etaL (1992) Brain Res. 593, 1-6 42 Prenen, G. H. M., Go, K. G., Postema, F., Zuiderveen, F. and Koff, J. (1988) Exp. Neurol. 99, 118-132 43 Yamasaki, Y., Kogure, K., Hara, H., Ban, H. and Akaika, N. (1991) Neurosci. Lett. 121,251-254 44 Boening, J. A., Kass, I. S., Cottrell, J. E. and Chambers, G. (1989) Neuroscience 33, 263-268 45 Tasker, R. C., Coyle, J. T. and Vornov, J. J. (1992) J. Neurosci. 12, 4298-4308 46 Burke, S. P. and Taylor, C. P. (1991) Soc. Neurosci. Abstr. 17,
1267 47 Hansen, A. J. (1985) PhysioL Rev. 65, 101-148 48 Nedergaard, M. and Astrup, J. (1986) J. Cerebr. Blood Flow Metab. 6, 607-615 49 Gill, R., Andin6, P., Hillered, L., Persson, L. and Hagberg, H. (1992) J. Cerebr. Blood Flow Metab. 12, 371-379 50 Kass, I. S., Abramowicz, A. E., Cottrell, J. E. and Chambers, G. (1992) Neuroscience 49, 537-543 51 Haigney, M. C. P., Miyata, H., Lakatta, E. G., Stern, M. D. and Silverman, H. S. (1992) Circ. Res. 71,547-557
Tauprotein and the neurol brillarypathologyofAIzheimer's disease Michel Goedert
Michel Goedertis at the MedicalResearch Council,Laboratory of MolecularBiology, HillsRoad, Cambridge, UK CB22QH.
Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in an abnormally phosphorylated state. PHF-tau is hyperphosphorylated on all six adult brain isoforms. As a consequence, tau is unable to bind to microtubules and is believed to selfassemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues are involved in the abnormal phosphorylation of tau. Alzheimer's disease is characterized clinically by a progressive loss of memory and other cognitive functions, resulting in a profound dementia. The intellectual decline is accompanied by the progressive
Fig.
1. Neurofibrillary pathology in the entorhinal cortex. The section was stained with an anti-tau antiserum. Abbreviations: NFT, neurofibrillary tangle; NT, neuropil threads; NP, neuritic plaque. Scale bar, lO0t~m. (Reproduced, with permission, from Ref. 643 460
© 1993, ElsevierSciencePublishersLtd,(UK)
accumulation in the brain of insoluble fibrous material, both extracellularly and within nerve cells. Extracellular deposits are made of beta-amyloid protein A[~1'2. Initial deposits are non-fibrillar but are progressively transformed into fibrils, giving rise to the characteristic amyloid plaques. Neurofibrillary lesions constitute the intraneuronal deposits. They are found in cell bodies and apical dendrites as neurofibrillary tangles, in distal dendrites as neuropil threads and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (Fig. 1). Ultrastructurally, all three lesions contain abnormal paired helical filaments (PHFs) as their major fibrous components and straight filaments (SFs) as their minor fibrous components (Fig. 2)3. NeurofibriUary lesions develop in the nerve cells that undergo degeneration in Alzheimer's disease. Their relative insolubility enables them to survive after the death of the affected nerve cells as extracellular tangles (or ghost tangles) that accumulate in the neuropil. These are then engulfed by astrocytes and are probably slowly degraded. Over the past five years significant progress has been made in unravelling the molecular composition of PHFs and in deducing possible mechanisms that may lead to their assembly. Current evidence strongly suggests that they are made entirely of the microtubule-associated protein tan in an abnormally phosphorylated state. Moreover, earlier results 4 indicating that the extent and topographical distribution of neurofibrillary lesions provide a reliable pathological correlate of the degree of dementia have been confirmed and extended 5'6.
Neuropathological stages of Alzheimer's disease The development of the neurofibrillary lesions is not random but follows a stereotyped pattern with regard to affected cell types, cellular layers and brain regions, with little individual variation. This has recently been used to define six neuropathological stages of Alzheimer's disease (Fig. 3) 2. The very first nerve cells in the brain to develop neurofibrillary lesions are located in layer pre-alpha of the trans-entorhinal region, thus defining stage I. Stage II shows a more severe involvement of this region, as well as a mild involvement of the pre-alpha TINS, VoL 16, NO. 11, 1993
Na ÷ currents that fail to inactivate Charles P. Taylor non-inactivating Na + current (also called sustained or CharlesP. Taylorisat persistent Na + current) has been characterized by theDeptof sensitivity to TTX in a variety of neurons, including Neuroscience squid axon 1, cerebellar Purkinje cells2 (Fig. 1), Pharmacology,Parkeneocortical pyramidal cells (Fig. 2) 3-5, thalamic DavisPharmaceutical Research,Divisionof neurons 6, CA1 hippocampal pyramidal cells7'8, striatal Wamer-LambertCo., neurons 8 and mammalian CNS axons9. Sustained Na + 2800PlymouthRd, currents have also been observed in glial cells 1° and in AnnArbor, several other excitable cells (see the discussion in ~HI48105,USA. Ref. 11). Most sustained Na + currents are blocked by TTX, by internal application of the permanently charged local anesthetic QX-314 or by application of solutions with reduced Na + concentration, and they are mostly insensitive to Ca 2+ channel blockers such as Ba2+, Co 2+, Mn z+ or Cd 2+. Sustained Na + current is typically a very small fraction of the peak inward current, and it has often been ignored in formal analyses such as the original study of Hodgkin and Huxley1~. In addition to the sustained current that is the subject of most of this review, there is a sustained Na + current predicted by the Hodgkin-Huxley analysis that occurs because the voltage dependence of Na + current activation and inactivation overlap. In contrast to non-inactivating current, this 'window current' only occurs over a narrow range of membrane voltages between about -50 and -40mV. In squid axon I and in adult mammalian pyramidal cells 3'4'7 non-inactivating Na + current represents 1-3% of the
Textbook accounts give the impression that Na + channels are short-acting binary switches: depolarization opens them, but only for about one millisecond. In contrast to this simplified view, a small but significant fraction of the total Na ÷ current in neurons occurs because channels open after long delays or in longduration bursts of openings. Such non-inactivating Na + current acts physiologically in neurons to amplify synaptic potentials and enhance endogenous rhythmicity, and also to aid repetitive firing of action potentials. In glial cells it also may regulate Na+-K + ATPase activity. The evidence for non-inactivating Na + current in a variety of neurons and glia is reviewed, along with a brief discussion of its ion channel substrate and its relevance for neurological diseases and drug therapy. Voltage-sensitive Na + channels are one of the basic building blocks of the nervous system, since they allow action potentials to propagate long distances down axons by causing regenerative and transient increases in Na + ion permeability. Several different classes of voltage-sensitive Na + channels have been identified, and some of these differ in their kinetics such as the rate of activation or inactivation or their sensitivity to the specific blocker tetrodotoxin (TTX). In contrast to differences in kinetics of various Na ÷ currents, a small fraction of Na ÷ current fails to inactivate even with prolonged depolarization. Such
A
B Mg2+, TTX
20 mV 2 nA i
I __
|
L-. •
,
-- L
:
I~
100 ms
Fig. 1. (A) Na+-dependent plateau potentials recorded by sharp electrode current-clamp from a Purkinje neuron in guinea-pig2+ cerebellar, tissue slice.2 Tissue was bathed with solution containing " /Hg2 + (3.7m/v1) and no Ca2 + to block Ca currents. Supenmposed traces show the results of transmembrane current pulses (150ms duration, ~ O. 17 nA, 0.31 hA, 0.42 nA, 0.82 nA and 2.0 nA) from the recording electrode. Rapid Na +-dependent action potentials are seen, but some traces also show plateau potentials of about 20 mV that outlast the applied current by up to 350ms. (B) Rapid action potentials and plateau potentials are abolished by application of tetrodotoxin (TTX) (301~). Note that the time constant to charge the membrane passively is approximately 30 ms, which is a much shorter period of time than the duration of plateau potentials. These findings indicate that plateau potentials are caused by a non-inactivating current from voltage-sensitive Na + channels. TINS, Vol. 16, NO. 11, 1993
© 1993, Elsevier SciencePublishersLtd, (UK)
455
A
:
B
:
C
b
TTX
c
Fig. 2. Persistent Na + current was recorded from pyramidal tract neurons using a sharp intracellular microelectrode in slices of cat neocortex maintained in vitro 3. (A) Current-clamp records are compared with (B) voltage-clamp response to a slowly rising voltage ramp in the same neuron. In both parts, voltage is shown by the upper traces and current by the lower traces. Both (,4) and (B) show evidence of sustained Na + current. In (,4) transmembrane voltage responses are shown in response to three different amounts of applied current (trace 1, -O.33 nA; trace 2, +O.33 nA and trace 3, +O.67nA, each lasting for 200ms). Note that action potentials in trace 3 are preceded by a slower depolarizing potential (between lines b and a) that traverses the voltage range corresponding with a region of negative slope in the voltage-clamp record [inward rectification in (B)]. Line d indicates the resting membrane voltage (-75mY). (C) The negative slope region of voltage-clamp records is made finear by appfication of 11ZMtetrodotoxin (TTX), indicating that it is caused by voltage-sensitive Na + channels. Other experiments (not shown) demonstrate that the negative slope region is not altered by 2 mM COCI2, but is blocked by intracellular appfication of the local anesthetic QX-314.
peak amplitude of Na+current. Sustained current has sometimes been designated lNa p or INa (slow)because it persists for long periods of time, in contrast to the widely studied transient current. These designations imply that sustained current is due to activation of a separate class of Na + channels than those responsible for transient current, but recent single-channel studies (see below) argue against that view. The regenerative nature of sustained Na + currents explains long-lasting plateau potentials 2'4'8'13 (Fig. 1) and sub-threshold depolarizations 14 that are particularly prominent if repolarizing K + currents are blocked. Such Na+-dependent plateau potentials can last for seconds, much longer than the passive membrane-charging time constant. Thus, plateau potentials are caused by an inward current that fails to inactivate even with very long durations of depolarization. Sustained Na + current is responsible for inward rectification that causes a negative slope region in steady-state I-V curves in electrophysiological studies of neocortical pyramidal cells 3'4 (Fig. 2B) and hippocampal neurons 15. Such inward rectification is reduced by TTX or by intracellular application of the local anesthetic QX-314. As with rapidly inactivating Na ÷ current, sustained current is
A
regenerative: as channels become activated, the membrane depolarizes and additional channels are recruited. Recent studies 8 have revealed an extremely slowly activating and de-activating component of sustained inward current in isolated stfiatal and hippocampal neurons that is TTX-insensitive (Fig. 3). However, in agreement with studies of rapidly inactivating Na + current, the slow current is blocked by replacement of Na + ions with Tris ions and it is partly supported when Na + ions are replaced by K + ions. Nevertheless, the insensitivity of this current to "FI'X indicates that it is different from conventional Na ÷ currents, and this may be due to the action of a different type of Na + channel protein.
Ion channels responsible for sustained Na + current Several previous studies did not determine whether sustained Na + current is caused by a novel type of Na + channel or by the same type of channel responsible for transient Na + current 4,7. Recently, the single-channel analysis by Alzheimer et al. n has shown that membrane depolarization can cause three types of Na + channel gating in a single small
B 90 mM Na ÷
C
0 mM Na +, 0 mM K +, 90 mM Tris
90 mM K +
4oopr 90 ms
Fig. 3. Voltage-clamp records from isolated striatal neurons in vitro 8 show (A) a slowly activating inward current that is (B) blocked by replacement of Na + by Tris ions and is (C) partly supported in medium with 90mM K +. Experiments were performed in the presence of 40 mM tetraethylammonium, 4 mM 4-aminopyridine, 3 m M Mn 2+, no added Ca2+ and 12 ~ tetrodotoxin (TTX) to block K +, Ca2+ and rapid Na + currents. Insensitivity to TTX suggests that these sustained and kinetically very slow Na + currents are caused by a different kind of Na + channel than those causing currents shown in Figs 1 and2. Note that the peak ampfitude of these currents (approximately 3OOpA) is much less than those of rapidly inactivating Na + currents. 456
TINS, Vol. 16, No. 11, 1993
2.0 m V membrane patch of acutely isolated neocortical neurons (Fig. 4). In addition to familiar short-latency RMP -'~----_.j ........................ ". . . . . . . . . . . . . . . . . . . . . . . . . transient openings following a depolarizing step, Na + - 5 0 m V channels can display infrequent brief openings that occur at relatively long delays after the start of depolarization. These 'delayed, brief openings' have durations similar to those of 'normal, rapid' Na ÷ current. The delayed brief openings probably account for a significant fraction of sustained current observed in whole-cell recordings. In the same patch of membrane that displays mostly transient Na + channel openings, about 1% of traces show very prolonged bursts of openings lasting tens of milliseconds or more (Fig. 4). These sustained bursts sometimes appear in 'blocks' or 'runs' that last for seconds. Similar prolonged bursts appear more commonly in cloned rat brain type-III Na ÷ channels that are physiologically expressed mainly in the immature brain TM. Prolonged bursts in both preparations have identical single-channel conductances and similar voltage dependence for activation to those observed for brief openings. In patches from acutely isolated brain cells u, the mean open time for Na ÷ channels during a prolonged burst was found to be voltage-dependent (0.5 mS at -60mV and 30mS at -20mV). The mean open time for delayed brief openings was also voltage dependent, but only within a much narrower range of durations. Because sustained bursts have such long durations, and because individual openings are longer with large depolarizations, prolonged bursts may contribute significantly to sustained Na* current recorded from whole cells, despite their relatively infrequent occurrence. The simplest interpretation of the single-channel data is that an individual Na ÷ channel can open either i~ :~ "': "i %~--~- :-'; :r'~¢i ~ - c - : ~'~- ::r ":':::" :--"::-': ",; j- ~'~'" immediately after depolarization (short-latency brief openings) or with a lower probability after a delay (delayed brief openings), but the same channel occasionally enters an open state that lacks fast inactivation. An alternative explanation for sustained single-channel gating is that a different class of Na ÷ channel proteins or a sub-population of altered channels co-exist with 'normal' channels in patches. The latter hypothesis supposes that the altered channels are usually silent (and thus are not detectable), but that they occasionally open in a sustained manner in the same patch of membrane that contains mostly normal channels. This possibility seems unlikely because sustained Na ÷ channel openings have been observed in a variety of other tissues besides the brain, including skeletal and cardiac muscle (see discussion in Ref. 11). They have also been observed in mammalian cells transfected with cDNA for rat brain type-IIA Na + channels ~7 and in X e n @ u s oocytes injected only with rat brain type-III Na ÷ channel mRNA 16. On statistical grounds Moorman et al. 16 have rejected the hypothesis that there is a different population of channels for sustained openings (a single Fig. 4. Voltage-clamp recordings from a small celltype of Na + channel might be altered even if attached patch on an acutely isolated rat neocortical expressed from a single mRNA by, for example, post- neuron ~1 show that most depolarizations cause rapid, translational processing). The authors of both recent brief channel openings. Occasionally, delayed brief opensingle-channel studies 11 ' 1(5" favor the idea that sus- ings (fourth trace down) or delayed but very prolonged bursts of openings (third trace from bottom) occur. All of tained openings occur interspersed with short- these types of channel openings are sensitive to tetroduration openings because of 'modal' gating, meaning dotoxin (not shown). Prolonged bursts and delayed brief occasional transitions of Na ÷ channels to a confor- openings may both contribute to the sustained currents mational state from which rapid inactivation is un- seen in whole-cell recordings such as those of Figs 1 and likely. 2. Abbreviation: RMP, resting membrane potential
TINS, Vol. 16, No. 11, 1993
457
(1)
investigated. In cardiac cells, application of lysophosphatidylcholine (a metabolite of phospholipid breakdown) greatly enhances the fraction of sustained Na + current 2°, although the mechanism of this effect has not been established. Additional experiments will no doubt improve our knowledge of the mechanisms that modulate Na + channel gating to enhance or reduce sustained current.
Na +
[Na+ ]i [K+ ]i
-
K+ or
+
P h y s i o l o g i c a l f u n c t i o n of n e u r o n a l n o n - i n a c t i v a t i n g Na + c u r r e n t s
EPSPs, or responses to iontophoretic glutamate application in hippocampal pyramidal cells, increase in size when they occur within the voltage range of sustained Na + currents 2z'22. Thus, relatively slow synaptic potentials that otherwise would not elicit action potentials become effective. The amplification of sub-threshold depolarizations is greatly reduced by K+ TTX or by the intracellular administration of QX-314 Fig. 5. Model for Na + channel modulation of Na+-K + (Refs 21, 22). In photoreceptor cells and certain other ATPase activity in glial cells2e. Na + channels maintain neurons without prominent action potentials, nonintracellular [Na +] at levels that provide a substrate for Na +-K + exchange. Tetrodotoxin blocks Na + channels (1) inactivating Na + currents amplify small changes in and causes intracellular [Na +] to decrease to very low membrane potential 23. levels, inactivating Na+-K + exchange. In contrast, In the presence of sustained Na + currents, repetistrophanthidin and ouabain directly inhibit the A TPase tive action potentials are more likely and threshold protein (2). for regenerative action is set primarily by sustained Na + current 3'6'22. Thus, the sustained 'sub-threshold' Several studies 4'5'7'8'11 suggest that sustained Na + current may be a major determinant of the rate of currents occur on or close to pyramidal cell bodies. repetitive firing of action potentials. It could also be Thus, non-inactivating Na + current might be es- speculated that sustained Na + currents assist passive pecially prominent in type-I Na + channels that are current flow from one part of neuronal membrane to localized primarily on somata and near the primary another and thus spatially integrate synaptic potenbranches of large dendrites TM. However, voltage- tials, as has been proposed for sustained dendritic clamp experiments with cloned type-I Na + channel Ca2+ currents 24. proteins show rapid inactivation that is at least grossly Neocortical pyramidal cells display endogenous similar to that of type-IIA channels (Scheuer, T. and depolarizations that cause rhythmic firing of action Catterall, W. et al., pers. commun.). Non-inactivating potentials and that are sensitive to TTX 5,25. Thus, Na + currents are also present in peripheral nerves, non-inactivating Na + channels may cause endogenous where type-I Na + channels are rare 9. rhythmicity at about 7Hz, near the frequency of Most of the analyses discussed above are consist- 'theta' rhythm in EEG recordings. Sustained Na + ent with the idea that sustained Na + currents arise currents are one of a fairly large family of voltagefrom infrequent sustained gating of completely activated ionic currents that contribute to oscillations 'normal' Na + channels. However, the recent descrip- in membrane potential independent of action potention s of a slow TTX-insensitive Na + current in tials24. neurons of the mammalian brain indicates that at least a fraction of sustained current in some neurons is from N o n - i n a c t i v a t i n g Na + c u r r e n t s in glial cells a distinct population of ion channel proteins (see Recent work by Sontheimer and co-workers 26 above). suggests that non-inactivating Na + channels play an important role in gila, where action potentials do not M o d u l a t i o n of s u s t a i n e d Na + c u r r e n t s occur physiologically (and therefore rapidly inactiSustained Na + currents might be modified or vating currents are mostly irrelevant). Glia possess modulated by phosphorylation of channel proteins, voltage-dependent Na + channels, including a novel post-translational processing of channel proteins or by type that is similar in many respects to those in other intracellular messengers. Phosphorylation of neurons 27, although the total membrane conductance Na + channels by protein kinase C slows the time- is dominated by K + channels. One class of glial Na + course of Na + channel inactivation and makes sus- channel proteins is significantly less sensitive to TTX tained bursts of openings more common 17, but it has than neuronal channels. Electrophysiological studies not been established whether the fraction of non- show that sustained Na + currents are present in glial inactivating current in whole-cell recordings is in- cells 1°'2a, and sustained currents are the only ones creased by this treatment. Proteases such as trypsin expected under physiological conditions. or papain practically abolish rapid inactivation of Na + Treatment of glia in cell cultures with TTX causes channels when applied by an intracellular micro- slow membrane depolarization and prevents Rb + pipette, leaving an unusually large fraction of non- influx via the Na+-K + ATPase 26 (see Fig. 5). These inactivating Na + current 19. Endogenous cytoplasmic effects of TTX mimic but do not add to those of proteases might also cleave the inactivation gate of ouabain or strophanthidin (specific ATPase inhibitors). Na + channel proteins and thereby increase sustained It is proposed that the effects of TTX in glia stem Na + current, but this hypothesis has not been from loss of electrogenic Na+-K + pumping. The 458
TINS, VoL 16, No. 11, 1993
inhibition of ATPase activity by TTX is interpreted to mean that an inward 'leak' of Na + through voltagesensitive channels is necessary to allow the N a + - K + ATPase to exchange Na ÷ ions for external K + ions. When Na + influx is blocked with TTX, action of the Na+-K + pump lowers intracellular [Na +] so much that further exchange of the two ions can no longer occur, and the transmembrane K + gradient eventually decays. Sustained TTX application kills glial cells, presumably by inactivating the ATPase. Therefore, non-inactivating Na + channels in glia may provide the correct intracellular [Na+] for gila to perform their K+buffering function in brain tissues. It is further speculated26 that activation of Na + channels may increase glial Na+-K + ATPase activity by raising cytoplasmic [Na +] in response to glial depolarization. Anticonvulsants
The drugs phenytoin and carbamazepine interact with Na + channels in much the same way as lidocaine, by reducing the probability that Na + channels can open from normal resting potentials ~9'3°. When fast inactivation is removed by treatment with proteolytic enzymes, phenytoin still reduces the probability of channel opening at low micromolar concentrations 31, and so phenytoin and similar drugs are expected to inhibit sustained Na + currents. Results with cardiac myocytes32 also indicate that lidocaine mimics or stabilizes the 'slow inactivated' state of Na + channels. It will be interesting to determine whether anticonvulsants and local anesthetics inhibit sustained neuronal Na + currents at lower concentrations than they inhibit rapid Na + currents, as already demonstrated with local anesthetics in cardiac myocytes33 and with other cardiac Na + channel modulators such as the experimental compound R 56865 (Ref. 34) or with intraceUular application of QX-314 to neurons 4. Relevance
to ischemic
stroke
Several drugs known to modulate Na + channels prevent neuronal damage in models of brain ischemia. Although convincing evidence is still lacking, a variety of data suggest that sustained Na + currents might be involved. Neuroprotective Na+-modulating drugs include lidocaine 35-a7 and phenytoin36'38'39, as well as newer agents such as RS-874764° and BW-1003C8741. These compounds undoubtedly act at multiple sites, but each of them potently modulates voltagedependent Na + channels. In contrast to these drugs, TTX is very selective for Na + channels and it also prevents neuronal d a m a g e 36'37'42-45 and glutamate release 46 from hypoxia or ischemia in brain tissues. Neuroprotection with Na+-modulating drugs at concentrations that do not block action potentials 36'37 implies that sustained Na + currents may be involved, and sustained currents are known to be present in optic nerve 9 and in other CNS neurons'3 5 7' 8' where neuroprotection has been demonstrated. Other data also suggest that sustained Na + currents contribute to ischemic damage. Long bursts of action potentials that might contribute significant Na + influx during ischemia have not been described, but sustained depolarizations that would particularly favor non-inactivating Na + currents are often seen 47-49 . A m a s s i v e T T X - s e n s i t i v e Na + accumulation occurs in brain slices deprived of o x y g e n in vitro, and this Na + influx continues e v e n after action potentials c e a s e 5°. TINS, Vol. 16, No. 11, 1993
Such a large TTX-sensitive influx of Na + ions would require very large numbers of action potentials, but even a relatively small Na + current sustained over minutes could contribute a relatively large flux of Na + ions. A similar hypothesis for the role of noninactivating Na + current in ischemic damage has been advanced for cardiac myocytes5z, where a large fraction of toxic Caz+ entry appears to follow from increased cytosolic Na + concentration.
Selected references 1 Gilly, W. F. and Armstrong, C. M. (1984) Nature 309, 448-450 2 Uin&s, R. and Sugimori, M. (1980) J. Physiol. 305, 171-195 3 Stafstrom, C. E., Schwindt, P. C. and Crill, W. E. (1982) Brain Res. 236, 221-226 4 Stafstrom, C. E., Schwindt, P. C., Chubb, M. C. and Crill, W. E. (1985) J. NeurophysioL 53,153-170 5 Alonso, A. and Llin,~s, R. (1989) Nature 342, 175-177 6 Jahnsen, H. and Llin&s, R. (1984) J. Physiol. 349, 227-247 7 French, C. R., Sah, P., Buckett, K. J. and Gage, P. W. (1990) J. Gen. Physiol. 95, 1139-1157 8 Hoehn, K., Watson, T. W. J. and MacVicar, B. A. (1993) Neuron 10, 543-552 9 Stys, P. K., Sontheimer, H., Ransom, B. R. and Waxman, S. G. Proc. Natl Acad. 5ci. USA (in press) 10 Barres,B. A., Koroshetz, W. J., Chun, L. L. Y. and Corey, D. P. (1990) Neuron 5, 527--544 11 Alzheimer, C., Schwindt, P. C. and Crill, W, E. (1993) J. Neurosci. 13,660-673 12 Hodgkin, A. L. and Huxley, A. F. (1952) J. PhysioL 117, 500-544 13 Jahnsen, H. (1986) J. Physiol. 372, 129-147 14 MacVicar, B. A. (1985) Brain Res. 333,378-381 15 Hotson, J. R., Prince, D. A. and Schwartzkroin, P. A. (1979) J. NeurophysioL 42,889-895 16 Moorman, J. R., Kirsch, G. E., VanDongen, A. M. J., Joho, R. H. and Brown, A. M. (1990) Neuron 4, 243-252 17 Numann, R., Catterall, W. A. and Scheuer,T. (1991) Science 254, 115-118 18 Westenbroek, R. E., Merrick, D. K. and Catterall, W. A. (1989) Neuron 3,695-704 19 Gonoi, T. and Hille, B. (1987) J. Gen. Physiol. 89, 253-274 20 Undrovinas, A. I., Fleidervish, I. A. and Makielski, J. C. (1992) Circ. Res. 71, 1231-1241 21 Deisz, R. A., Fortin, G. and Zieglg~nsberger, W. (1991) J. Neurophysiol. 65, 371-382 22 Hu, G-Y. and Hvalby, O. (1992) Exp. Brain Res. 88, 485-494 23 Vallet, A, M., Coles, 3. A., Eilbeck, J. C. and Scott, A. C. (1992) J. Physiol. 456, 303-324 24 Llin,hs,R. (1988) Science 242, 1654--1664 25 Silva, L, R., Amitai, Y. and Connors, B. W. (1991) Science 251,432-435 26 Sontheimer, H., Fernandez-Marques, E., UIIrich, N. and Waxman, S. G. (1993) 5oc. Neurosci. Abstr. 19, 688 27 Gautron, S. et aL (1992) Proc. Natl Acad. Sci. USA 89, 7272-7276 28 Howe, J. R. and Ritchie, J. M. (1992) J. PhysioL 455,529-566 29 Catterall, W. A. (1987) Trends PharmacoL Sci. 8, 57-65 30 Ragsdale, D. S., Scheuer, T. and Catterall, W. A. (1991) 44ol. Pharmacol. 40, 756--765 31 Quandt, F. N. (1988) 44ol. PharmacoL 34, 557-565 32 Zilberter, Y., Motin, L., Sokolova, S., Papin, A. and Khodorov, B. (1991) J. 44ol. Cell CardioL 23 (Suppl. 1), 61-72 33 Ju, Y-K., Saint, D. A. and Gage, P. W. (1992) Br. J. PharmacoL 107, 311-316 34 Verdonck, F., Bielen, F. V. and Ver Donck, L. (1991) Eur. J. Pharmacol. 203, 371-378 35 Rasool, N., Faroqui, M. and Rubinstein, E. H. (1990) Stroke 21,929-935 36 Taylor, C. P. and Weber, M. (1992) Soc. Neurosci. Abstr. 18, 1135 37 Stys, P. K., Ransom, B. R. and Waxman, S. G. (1992) J. Neurophysiol. 67, 236--240 38 Clifton, G. L, Taft, W. C., Blair, R. E., Choi, S. C. and DeLorenzo, R. J. (1989) Stroke 20, 1545-1552 39 Boxer, P. A. et al. (1990) Stroke 21 (Suppl. III), 47-51 40 Alps, B. J. (1992) Br. J. Clin. Pharmacol. 34, 199-206
Acknow/edgernents I wishto thank BilICatterall, BrianMacVicar, HaraldSonb~eimer and PeterStys for providing unpublished manuscriptsand informab'on.Special thanks to David Rock andJeffery Kocsisfor helpful discussions and criticismof the manuscript. 459
41 Meldrum, B. S. etaL (1992) Brain Res. 593, 1-6 42 Prenen, G. H. M., Go, K. G., Postema, F., Zuiderveen, F. and Koff, J. (1988) Exp. Neurol. 99, 118-132 43 Yamasaki, Y., Kogure, K., Hara, H., Ban, H. and Akaika, N. (1991) Neurosci. Lett. 121,251-254 44 Boening, J. A., Kass, I. S., Cottrell, J. E. and Chambers, G. (1989) Neuroscience 33, 263-268 45 Tasker, R. C., Coyle, J. T. and Vornov, J. J. (1992) J. Neurosci. 12, 4298-4308 46 Burke, S. P. and Taylor, C. P. (1991) Soc. Neurosci. Abstr. 17,
1267 47 Hansen, A. J. (1985) PhysioL Rev. 65, 101-148 48 Nedergaard, M. and Astrup, J. (1986) J. Cerebr. Blood Flow Metab. 6, 607-615 49 Gill, R., Andin6, P., Hillered, L., Persson, L. and Hagberg, H. (1992) J. Cerebr. Blood Flow Metab. 12, 371-379 50 Kass, I. S., Abramowicz, A. E., Cottrell, J. E. and Chambers, G. (1992) Neuroscience 49, 537-543 51 Haigney, M. C. P., Miyata, H., Lakatta, E. G., Stern, M. D. and Silverman, H. S. (1992) Circ. Res. 71,547-557
Tauprotein and the neurol brillarypathologyofAIzheimer's disease Michel Goedert
Michel Goedertis at the MedicalResearch Council,Laboratory of MolecularBiology, HillsRoad, Cambridge, UK CB22QH.
Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in an abnormally phosphorylated state. PHF-tau is hyperphosphorylated on all six adult brain isoforms. As a consequence, tau is unable to bind to microtubules and is believed to selfassemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues are involved in the abnormal phosphorylation of tau. Alzheimer's disease is characterized clinically by a progressive loss of memory and other cognitive functions, resulting in a profound dementia. The intellectual decline is accompanied by the progressive
Fig.
1. Neurofibrillary pathology in the entorhinal cortex. The section was stained with an anti-tau antiserum. Abbreviations: NFT, neurofibrillary tangle; NT, neuropil threads; NP, neuritic plaque. Scale bar, lO0t~m. (Reproduced, with permission, from Ref. 643 460
© 1993, ElsevierSciencePublishersLtd,(UK)
accumulation in the brain of insoluble fibrous material, both extracellularly and within nerve cells. Extracellular deposits are made of beta-amyloid protein A[~1'2. Initial deposits are non-fibrillar but are progressively transformed into fibrils, giving rise to the characteristic amyloid plaques. Neurofibrillary lesions constitute the intraneuronal deposits. They are found in cell bodies and apical dendrites as neurofibrillary tangles, in distal dendrites as neuropil threads and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (Fig. 1). Ultrastructurally, all three lesions contain abnormal paired helical filaments (PHFs) as their major fibrous components and straight filaments (SFs) as their minor fibrous components (Fig. 2)3. NeurofibriUary lesions develop in the nerve cells that undergo degeneration in Alzheimer's disease. Their relative insolubility enables them to survive after the death of the affected nerve cells as extracellular tangles (or ghost tangles) that accumulate in the neuropil. These are then engulfed by astrocytes and are probably slowly degraded. Over the past five years significant progress has been made in unravelling the molecular composition of PHFs and in deducing possible mechanisms that may lead to their assembly. Current evidence strongly suggests that they are made entirely of the microtubule-associated protein tan in an abnormally phosphorylated state. Moreover, earlier results 4 indicating that the extent and topographical distribution of neurofibrillary lesions provide a reliable pathological correlate of the degree of dementia have been confirmed and extended 5'6.
Neuropathological stages of Alzheimer's disease The development of the neurofibrillary lesions is not random but follows a stereotyped pattern with regard to affected cell types, cellular layers and brain regions, with little individual variation. This has recently been used to define six neuropathological stages of Alzheimer's disease (Fig. 3) 2. The very first nerve cells in the brain to develop neurofibrillary lesions are located in layer pre-alpha of the trans-entorhinal region, thus defining stage I. Stage II shows a more severe involvement of this region, as well as a mild involvement of the pre-alpha TINS, VoL 16, NO. 11, 1993