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Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line.

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Amvotro~hcLateral Sclerosis
Immunoglobuhns Increase Ca2 Currents in
a Motoneuron Cell Line
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Dennis R. Mosier, MD, PhD," Pietro Baldelli, PhD,'i Osvaldo Delbono, MD, PhD,t$
R. Glenn Smith, MD, PhD," Maria E. Alexianu, MD," Stanley H. Appel, MD," and Enrico Stefani, MD, PhD4'1"
~
The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing
progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients
with sporadic ALS have serum antibodies that bind to purified Gtype voltage-gated calcium channels and that antibody
titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been
found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using wholecell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown
to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the
polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca" currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels
may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.
Mosier DR, Baldelli P, Delbono 0, Smith RG, Alexianu ME, Appel SH, Stefani E.
Amyotrophic lateral sclerosis immunoglobulins increase Ca' ' currents in a
motoneuron cell line. Ann Neurol 1095;37:102- 109
Amyotrophic lateral sclerosis (ALS) is a progressive
and eventually lethal disease that produces degeneration of cortical and spinal motoneurons [ I]. Although
some cases of the familial form of ALS have been
linked to alterations in the gene for Cu' /Zn'+ superoxide dismutase f2, 31, the cause of the sporadic form
of ALS, which represents 90% of reported cases, remains unclear. The increased incidence of autoimmune
disorders [4] and paraproteinemias IS] in patients with
ALS has furnished clinical evidence for altered immunity in this disorder. Experimental evidence continues
to provide increasing support for a role of autoimmune
factors in the pathogenesis of ALS [I, 6-10). Immunemediated destruction of upper and lower motoneucons has been demonstrated in an animal model, experimental autoimmune gray matter disease [6]. The
presence of IgG in motoneurons as well as infiltrates
of T lymphocytes and microglia in spinal gray matter
and motor cortex has been reported in patients with
ALS [7, 81. Recently, serum antibodies binding to purified L-type voltage-gated calcium channels have been
demonstrated in 75% of patients with sporadic ALS,
without evidence of such antibodies in patients with
familial ALS [9]. Passive transfer of immunoglobulins
from ALS patient sera has been shown to increase
spontaneous quantal transmitter release from mouse
motor nerve terminals [lo].
While the results of the passive transfer studies were
hypothesized to result from increased intracellular
Ca' following an ALS IgG-mediated enhancement of
Ca' currents, an inhibition of Ca'+ current was noted
when L-type voltage-gated Ca" channels from skeletal
muscle were tested with ALS immunoglobulins [ 1I].
However, when neuronal P-type Ca" channels were
exposed to ALS immunoglobulins, an enhancement of
Caz+ current was observed f12). To better understand
the specific actions of ALS immunoglobulins on cell
types that are clinically affected in ALS (motoneurons),
we have produced a motoneuron hybrid cell line
(VSC4.1) by somatic fusion of N18TG2 neuroblastoma cells with rat embryonic ventral spinal neurons
C13-153. As has been shown for other motoneuron
hybrid lines 116, 171, differentiated VSC4.1 cells exhibit many properties of motoneurons, including cyclic AMP (CAMP)-inducible choline acetyltransferase
activity as well as voltage-gated Caz+ channels [IS}.
From the Departments of "Neurology and t Molecular Physiology
and Biophysics, Baylor College of Medicine, Houston, TX 77030.
Address correspondence ro Dr Mosier, Department of Neurology,
Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030.
Received Apr 19, 1994, and in revised form Jun 21. Accepred for
publication Aug 3, 1994.
fPresenr address: Department of Physiology and Pharmacology,
Bowman Gray School of Medicine, Winston-Salem, NC 27157.
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102 Copyright 0 1995 by the American Neurological Association
Differentiated VSC4.1 cells are also susceptible to
Ca2 -dependent cell death induced by ALS immunoglobulins [ls]. I n this study, w e have used electrophysiologic techniques to study t h e effects of immunoglobulins from ALS patients on Ca2+currents expressed in
the VSC4.1 motoneuron line and have documented a
relatively specific enhancing effect of ALS immunoglobulins on Ca2+ currents that are sensitive to t h e
polyamine funnel-web spider toxin (FTX) but insensitive to o-conotoxin GVIA (0-CgTx G V I A ) and wagatoxin IVa (o-Aga-IVa). Such information could
help determine whether ALS immunoglobulins, interacting with voltage-gated Ca2 channels, could increase
Ca” entry and provide a signal for subsequent injury
in susceptible cells, as has been suggested 118, 191.
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Materials and Methods
Preparation and Culture of VSC4.1 Cells
Preparation and culture of the parental cells (mouse
N 18TG2 neuroblastoma cells and embryonic day 15 rat ventral spinal neurons), somatic cell fusion, and hybrid selection
were performed as previously described [14, 151. Cells
from the resulting hybrid line, VSC4.1, were maintained in
log-phase growth in Dulbecco’s modified Eagle medium
(DMEM)/F12growth medium (GIBCO) supplemented with
Sato’s N 1 components [201 and 25% heat-inactivated newborn calf serum (Hyclone). Cells from passages 5 to 12 were
differentiated for 7 days in 1 mM dibutyryl-CAMP, with 0.4
kg/ml aphidocolin (Sigma) added 24 hours after initiating
exposure to CAMP, to inhibit the proliferation of cells unresponsive to CAMP-induced differentiation. Differentiated
cells were harvested by trituration following 1-hour incubation at 37°C in Ca2+/Mg2+-freeHanks’ balanced salt solution
containing 1 mM EDTA, and replated onto glass coverslips
precoated with poly-L-ornithine and fibronectin, at a density
of 20,000 cells per coverslip. Electrophysiologic recordings
were typically performed 3 to 7 days after replating, at a time
when Ca” current expression was maximal (Mosier DR,
Baldelli P, unpublished data). During this time, current expression in VSC4.1 cells was consistently two- to threefold
greater than expression of similar currents in N18TG2 cells,
when differentiated under identical conditions (Mosier DR,
Baldelli P, Delbono 0, et al, manuscript in preparation).
Patient Selection
Seven patients given a diagnosis of sporadic ALS were randomly selected and evaluated on the basis of medical history,
physical examination, electromyography, muscle biopsy, and
exclusionary clinical and biochemical studies [4]. None of
the ALS patients had evidence of multifocal motor conduction block, antiganglioside antibodies, or diabetes mellitus.
In addition, 2 patients with nonautoimmune neurologic illnesses, and 5 patients with autoimmune neurologic diseases
such as myasthenia gravis or autoimmune polyneuropathy,
were used as control patients. One patient with the LambertEaton myasthenic syndrome (LEMS), a disease known to exhibit autoantibodies against voltage-gated Ca2+ channels
1211, served as an additional control.
Inzmunoglobulin Preparation
Immunoglobulin fractions were purified from sera of ALS
and control patients by ammonium sulfate precipitation, ionexchange chromatography, and filtration dialysis as previously described [91, resulting in IgG preparations of 909%or
greater purity. IgG samples were stored under liquid nitrogen and dialyzed into HEPES-buffered sodium methanesulfonate or tetraethylammonium (TEA) methanesulfonate solutions prior to use. For electrophysiologic studies, IgGs
were locally added to the recording chamber to obtain final
bath concentrations ranging from 0.1 to 1.0 mg of protein/
ml. (To minimize the likelihood of a type I1 error, this concentration range was chosen to be up to 10-fold greater than
the IgG concentrations that have been shown to induce maximal levels of cell death in similarly differentiated VSC4.1
cells 1151.) These concentrations of IgG produced no immediately observable toxic effects on VSC4.1 cells (e.g., cell
swelling, increased leak current) following bath addition.
Electvophysiologic Recording Techniques
Voltage-gated Ca2 currents were measured in VSC4.1 cells
with the whole-cell configuration of the patch-clamp technique [22), using Ba2+ as the charge carrier. Coverslips
plated with differentiated VSC4.1 cells were placed in a specially prepared chamber mounted on the stage of an inverted
microscope (World Precision Instruments). The perfusion
chamber was connected by a KCI-filled agar bridge to a well
connected to ground by an Ag/AgCl wire. External solutions
typically contained (in mM), as follows: BaC12 40, TEA-CI
107.5, HEPES 10, tetrodotoxin 0.001 (pH 7.4) at 21°C to
23°C. In a few experiments, reductions in external Ba” concentration (to 5 or 10 mM) were compensated by isosmotic
substitution with TEA-CI. Recordings were performed with
fire-polished borosilicate pipettes of low resistance (0.5- 1.5
MO) filled with internal solution containing (in mM), as follows: cesium methanesulfonate 126, MgClz 4.5, Mg-ATP 4,
glucose 9, EGTA 9, HEPES 9 ( p H 7.4). All solutions were
filtered through a 0.45-km Millipore filter prior to use.
Seal resistances and capacitative transients were monitored
during experiments by applying small ( 5 mV) voltage pulses
superimposed on the command potential. The pClamp software version 5.5.1 and a TL-1 interface (Axon Instruments)
were used together with an IBM-compatible computer (486)
for generation of command potentials and for data analysis.
Currents were recorded with an Axopatch 200 integrating
patch clamp (Axon Instruments). Inward currents were
evoked by step depolarizations of 50- to 500-msec duration,
applied at a rate of 0.3 to 2.0 per minute. Voltage steps
were typically applied from a holding potential of - 50 mV.
Passive currents were subtracted on-line by negative small
(1/4 amplitude) subpulses from a subpulse holding potential
of -90 mV.
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Drugs and Toxins
Purified ETX (p-ETX), a polyamine toxin purified from funnel-web spider (Agelenopsis aperta) venom, and a synthetic
analogue, s-FIX E231, were obtained as a gift from D r R.
Llinks (New York University). Isradipine (PN200-110) was
purchased from Calbiochem. w-CgTx GVIA was purchased
from Peninsula Laboratories. w-Aga-IVa, a purified peptide
Mosier et al: ALS IgG Increase Motoneuron Ca“
Currents
103
Amyotrophic Lateral Sclerosis and Control Patient Data
Patient No.
Diagnosis
Sex
ALS
ALS
ALS
F
M
F
ALS
ALS
ALS
M
M
M
11
12
13
Normalh
AD
APN
GBS
MG
APN
MG
F
M
M
F
M
M
F
14
EMS'
M
1
2
3
4
5
6
7
8
9
10
Age
(yr)
Percent Increase in I,,,
Range (no. of cells)
65
58
51
36
70
49
-5.1 to +54.7 ( 8 )
+7.4 to +65.9 ( 6 )
- 1.9 to + 14.5 ( 4 )
-0.2 to +7.1 ( 4 )
-0.6 to + 14.2 ( 4 )
-0.5 to + 15.6 ( 4 )
51
77
p 0.01
p -:
0.02
p 0.002
:<
:e
ns.
p
p
:<
:<
0.001
0.002
- 11.6 to + 1.4 ( 5 )
to +2.7 (3)
42
54
35
+0.6
-8.8
-1.9
-2.1
-4.2
-6.9
54
-0.1 to +38.9 ( 2 )
49
66
Significancea
(1)
to
+3.7 ( 3 )
to +2.8 (3)
to +6.1 ( 4 )
to -0.1 ( 3 )
Amyotrophic lateral sclerosis (ALS) and control patients were matched for age (mean t SEM; ALS, 54.8 5 5.0 yr; controls, 53.4 rt_ 5.8 yr)
and sex. None of the ALS patients exhibited multifocal motor conduction block, detectable levels of antiganglioside antibodies, (or diabetes
mellitus. Effects of immunoglobulins on Ba2+ currents are presented as per Figure 3 legend.
"Statistical significance of responses to individual ALS immunoglobulin preparations over control responses were evaluated using one-tailed
Student's t tests, corrected for unequal variance.
b N o evidence of neurologic disease; however, the patient had a psychiatric diagnosis of Munchausen's syndrome.
'Without evidence of associated malignancy to date.
A D = Alzheimer's dementia; APN = autoimmune polyneuropathy; GBS = Guillain-Barre syndrome; M G
Lambert-Eaton myasthenic syndrome.
toxin from Agelenopsis aperta venom, was obtained from Peptide Institute, Inc. o-Conotoxin MVIIC (o-CgTx MVIIC)
was purchased from Research Biochemicals. S( - )BAY-K
8644 (Research Biochemicals) dissolved in 100% ethanol
was applied under conditions of near darkness due to its
known photosensitivity. Addition of equivalent amounts of
vehicle in the absence of toxin had no effect on Ba'+ currents. The activity of purchased toxins or drugs was verified
by measuring their effects on BaZt currents using whole-cell
patch clamp of neonatal rat dorsal root ganglia (DRG) neurons, or voltage-clamped XenopuJ oocytes injected with rat
brain messenger R N A 1241, under bath conditions similar to
those described above.
Mathematical and Statistical Analyses
Ba2+ currents were corrected for rundown in all ALS and
control experiments by linear extrapolation of current amplitudes measured prior to addition of immunoglobulin fractions. Linear extrapolation did not cause apparent overestimation of Ba2+ current responses after adding buffer solutions
without immunoglobulin (data not shown) or control immunoglobulin fractions (Table). Regression analyses were performed using least-square fits by in-house software. Statistical
significance of differences between means was evaluated using one-tailed Student's t tests, corrected if necessary for
unequal variance. Unless otherwise stated, data are presented
asmean ? SEM.
104 Annals of Neurology
Vol 37
N o 1 January 1995
=
myasthenia gravis; LEMS
=
Results
Calcium currents in CAMP-differentiated VSC4.1 cells
were typically measured using 40 mM Ba" as the
charge carrier, as described in Materials and Methods.
Inward current amplitude varied with the concentration of Ba" in the external solution, was unaffected
by isosmotic replacement of external C1- with methanesulfonate anion, and was blocked completely with 200
to 300 FM Cd2+ (Fig lA), consistent with inward currents resulting from Ba2+ influx through Ca'! channels. In 40 mM Ba2+, inward currents activated near
a potential of - 40 mV, displayed peak amp11tudes at
potentials of - 10 to 0 mV, and exhibited rapid, voltage-dependent inactivation (Mosier DR, Baldelli P,
Delbono 0, et al, manuscript in preparation).
Since a polyamine toxin purified from FTX has been
shown to block Ca'+ currents at mammalian m.otoneuron terminals 1251, we tested the effects of F T X and
its synthetic analogue, s-FTX [23], on BaZCcurrents
expressed in VSC4.1 cells. s-FTX was found )toblock
these Ba2+ currents completely (>95%) at bath concentrations of 3 to 10 p.M (see Fig IB). This blocking
effect was easily reversed by a brief washout (1-3 min)
of toxin. FTX purified from venom of the funnel-web
spider, Agelenopsis aperta, also blocked Ba2 currents
+
tions of 0.1 to 1.0 mg of protein/ml (see Materials and
Methods), increased the amplitudes of Ba2+ currents
evoked by voltage steps in VSC4.1 cells (see Fig 2A).
Cd2+200 pM
As shown in Figure 2B, the peak of the currentvoltage relationship was not significantly shifted following addition of ALS immunoglobulins. Increases in
L
Ba2+ current amplitude were typically apparent 2 to 4
40 rns
A
Control
minutes after addition of ALS immunoglobulin fractions, with maximal responses attained 10 to 45 min-10mV
utes following immunoglobulin application. These ef-50 mv ____...1_____________ _______ _______________ ________________.......
I. 50 mV
fects persisted for the duration of the experiments (see
Fig 2C) and were observed in experiments where the
C
i
,
__
Ba2+
concentration was lowered or external C1- was
i\
F .
replaced with methanesulfonate anion. Immunoglobulin effects on Ba2+ currents were not accompanied by
alterations in passive membrane properties (see Materials and Methods) and were not reversible by brief
a
400
PA
washout (5-10 minutes). Addition of buffer solutions
B
40 rns
without immunoglobulin fractions had no effect on
BaZ+currents in VSC4.1 cells.
F i g 1 . (A) Blockade of inward currents in VSC4.1 cells by 200
Ba2+ currents tested after ALS immunoglobulin exCJM Cd2+. Currents were evoked by step depolarizations from a
holding potential N h i of - 5 0 mV. using 40 mM B h + as the
posure remained sensitive to s-FTX (2.5-5 pM) or
charge carrier. (Bi Sensitivity o f B h + currents in VSC4.1 cells
p-FTX addition (n = 2; see Fig 2D), consistent with
to a synthetic form of a polyamine toxin-a’eriieed from funnelthe hypothesis that ALS immunoglobulins may act
web spider wnom (s-FTX).As above, currents recorded in 40
to amplify the ETX-sensitive Ba2+ currents described
m M B d + were woked by step depolarizationsfrom a Vh of
above. To further examine the possibility that Ba”
-50 mV with (a) 0 pM. (b) 1.5 pM, and (cJ 3.0 pM syncurrent increases resulted from amplification of a
thetir FTX present in the bath solution.
small subpopulation of voltage-gated Ca2+ channels,
(-)PN200-110 (n = 6), w-CgTx GVIA (n = 6), and
w-Aga-IVa (n = 2) were added following exposure
to ALS immunoglobulins. At concentrations similar to
(see Fig 2D). However, bath concentrations of up to
those described previously, (-)PN200-110 and w4 pM w-Aga-IVa, a peptide toxin also purified from
Aga-IVa did not alter Ba2+ current increases induced
Agelenopsis aperta venom {26], had no effect (n = 3
by ALS immunoglobulins. Although w-CgTx GVIA
cells), suggesting that the observed Ba” currents in
appeared to cause a small decrease (11%) in peak Ba’
VSC4.1 cells may be pharmacologically as well as eleccurrent amplitude in 1 of 6 experiments following ALS
trophysiologically distinct from the FTX-sensitive P
immunoglobulin addition, this effect could not account
currents described in cerebellar Purkinje cells 126,271.
for the magnitude of the increase (50%) produced by
Further pharmacologic characterization of VSC4.1
the ALS immunoglobulins in that experiment. In the
Ba2+ currents showed no effects of the dihydropyriremaining 5 experiments, w-CgTx GVIA had no effect
dine (DHP) antagonist (-)PN200-110 (2-20 pM; 5
on the magnitude of Ba2+ currents following ALS imcells), or the DHP agonist ( - )BAY-K 8644 (1-4 pM;
munoglobulin addition. These findings support the hy4 cells). The N-channel antagonist w-CgTx GVIA (1-6
pothesis that increases in Ba2+ currents following ALS
pM) was likewise ineffective in altering Ba2 currents
immunoglobulin addition are due to an effect on the
in 6 of 6 cells using 40 mM external Ba2+,and in 3 of
FTX-sensitive population of Ca2+ channels, rather
4 cells using 5 to 10 mM external Ba” (a 12% decline
than on a small subpopulation of pharmacologically dein peak current amplitude was seen in a single experifined L-, N-, or P-type Ca2+ channels.
ment following the addition of w-CgTx GVIA). A synImmunoglobulin fractions from 5 of 6 patients with
thetic preparation of the Ca2+ channel antagonist wALS (characterized in the Table) increased Ba2+ curCgTx MVIIC, which has been shown to inhibit a
rents above baseline values in a total of 16 of 30 experipopulation of Ca2+ currents resistant to the effects
ments. As illustrated by the scatter plot in Figure 3,
of DHPs, w-CgTx GVIA, and w-Aga-IVa 1281, also
Ba2+ current responses of individual cells to ALS imdid not appear to affect Ba2+ currents expressed in
munoglobulins varied between - 5% and 66%. AlVSC4.1 cells (1 pM; 2 cells).
though immunoglobulins from some patients appeared
Immunoglobulin fractions purified from patients
with sporadic ALS, when applied at bath concentrato produce larger responses than others, considerable
Y
V
‘PXA”
,
L
L
+
+
+
Mosier et al: ALS IgG Increase Motoneuron Ca2+ Currents
105
0
0
0
=?
Z L
-100
20 In8
A
I
40
40
--I
L
-110
I
my
pmc
1.do0
= 'r
0
0
8
-2000
Y,
CMltrd
ALS IgG
0.
*
-
O%&'
'mW)
d 4b #,
65
do
Time (min)
B
D
Fig 2. Efiects of aniyotrophii-lateral sclerosis (ALS, irnmzinoglobd i n s on Ba" currents in VSC4.1 cells. ( A )Representative current tram recorded in 40 rnM B h +, shoiving an inrrease in
magnitude of the peak inu'ard current folloioing addition of0.j
mglml ALS IgG to the bath solution. The stimulation protocol
i s depicted aboue the current traces. (Bi Current-voltage (I-V)
relationship of Ba' currents recorded from a representatiee
VSC4.I cell before and after 0.3 mglnil ALS IgG addition.
shozcling the lack of a significant shift in the peak of the I-V relationship following an increase in current amplitude due t o
ALS IgG. Each point represents the current amplitude recorded
in 40 m M B h during step depolarizations applied in 10 mV
increments from a holding potential Nh) o f - 70 m y . (c)Representatit'e experiment shou'ing the time course of increaJ.es in
peak Ba'+ cuwetzt amplitude following addition of ALS immu-
noglobulins. Each point represents peak current recorded in 10
niM Bh' during single-step depolarizations to - 20 mV from
a V, of - 80 mV, before and after bath addition of 1.0 mglml
ALS IgG. In this experinzent. external CI- was isosmotically
replaced with methanesulfonate. (Di Representative experiment
shouing the lack of effect of the N-channel antagonist aconotoxin G V I A (CltCgTx GVIA; 1 pMi and the dihydropyridine antagonist PN200-I 10 (20 p.Mi following an ALS immunoglobulin-induced increasr in Bd current. Each dara point
represents peak cuwent recorded in 40 mM B h during single
voltage steps t o 0 mV from a v h of - 30 mV. Subsequent addition of C U J Osuccessive I :jOO (imllvol) dilutions of purtj'iedfunnel-web spider toxin ( F T X ;final dilution, 1 :2501 is j?d/owed
by progressive reduction o f Ba' ' current amplitude belw baseline Iwels. 'The point at which the seal began to deteriorate.
variation in Ba2+ current responses was noted even
within sets of data obtained using immunoglobulins
from individual ALS patients (see Table). This variability of responses to immunoglobulins obtained from individual patients was not explicable by differences in
cell batch or passage number and may reflect intrinsic
heterogeneity of the differentiated VSC4.1 cell population, as previously described in cytotoxicity [15] or
biochemical assays [29J.
Immunoglobulin fractions from 7 control patients
( 5 with autoimmune neurologic disorders and 2 with
nonautoimmune neurologic or psychiatric illnesses)
failed to increase Ba'+ current amplitudes in a total of
22 experiments (see Fig 3). However, immunoglobulins from a patient with E M S , a disease in whiich Ca2+
channel autoantibodies are also found 1211, increased
Ba" current amplitude in a single experiment. This
finding was not unexpected, since both ALS and E M S
immunoglobulins have been shown to bind voltagegated Caz+ channels in an enzyme-linked immunosorbent assay 191, and to induce death of CAMPdifferentiated VSC4.1 cells [lS].
+
106 Annals of Neurology
Vol 37 No 1 January 1995
+
+
70
c 3
c
Ot
E J 20
E $ j 10
O O
a n
0
I
*
+
.&
......... .................L1. ..............
...........
j
-10
..........
Immunoglobulins purified from patients with sporadic ALS increased VSC4.1 Ba2+currents above baseline in about one-half of tested cells. Since specific
binding for w-CgTx GVIA and o-Aga-IVa, as well as
inhibition of ALS I&-induced cytotoxicity by these
agents, has been observed in the VSC4.1 cell line I1 51,
an effect of ALS IgG on small populations of CaZC
channels sensitive to these agents was initially considered as an explanation for the observed Ba2+ current
increases. However, the macroscopic homogeneity of
FTX-sensitive Ca’ + currents in VSC4.1 cells, together
with the inability of inhibitors of a number of other
Ca2+ channel types to alter Ba2 current increases due
to ALS immunoglobulins, suggests that the observed
BaL+ current increases result from enhancement of
FTX-sensitive Ca’ channels, rather than amplification
of Ba” current through a small subpopulation of Ca’+
channels of another type.
Variability in the magnitude of VSC4.1 Ba” current
increases due to ALS immunoglobulins was noted between samples obtained from different ALS patients
and also between cells tested with immunoglobulins
from the same patient. While the former may represent
differences in targeted epitopes and antibody binding
affinity or efficacy, which have been described in patients with other autoimmune diseases 13 l}, the latter
could not be explained on the basis of differences in
electrophysiologic properties, cell batch, or passage
number. The macroscopic homogeneity of the observed Ba2+ currents argues against heterogeneity of
these channels as an explanation for the variability of
responses to ALS immunoglobulins. However, variability could arise from differences in Ca2+ channel
regulation, since intrinsic heterogeneity of the differentiated VSC4.1 cell population has been previously described [ 15, 291. Nevertheless, immunoglobulin-induced increases in VSC4.1 Ba2’ currents
were observable in the majority of ALS patients
tested.
The lack of observed Ba” current increases due to
immunoglobulins obtained from sera of patients with
a variety of autoimmune and nonautoimmune neurologic disorders suggests that the effect of ALS immunoglobulins is specific and not related to a general effect
of autoimmune illness or neuronal compromise. However, the increase in Ba” currents caused by immunoglobulins from a patient with LEMS, an illness in which
anti-calcium channel antibodies also occur 12 I}, emphasizes that distinctions between these two illnesses
cannot be made solely on the basis of the present assay
method. Although our previous studies have shown
binding to purified L-type Ca” channels [9} and cytotoxic effects on VSC4.1 cells I151 of both ALS and
LEMS IgG, immunoglobulins from patients with these
two diseases may be distinguished by the selective
blockade of cytotoxic effects of ALS IgG by w-CgTx
GVIA Il51, and by specific binding of ALS IgG to the
+
0
i
-
-20
ALS
I
I
LEMS A.I. Non-A.I.
Controls
F i g 3. Sratter plot showing the distribution of peak Ba‘
cur-
vent changes after addition of immunoglobulins obtained from
amyotrophic lateral sclevosis (ALS)and controf patients. Each
data point represents the response of peak BaZ currents recorded
from a single VSC4.I cell t o application of immunoglobulins
from a single ALS or control patient. Effects of ALS and control
itnmunoglobulin preparations are presented as a percent increase
of peak gal+ current amplitude oz’er baseline leziels measured
prior to adding immunoglobulins.Responses were signtjcantb
greater after treatment with ALS immunoglobulins than after
addition of immunoglobulins from autoimmune (A.1.)disease
controls ( p < 0.0005) or nonautoimmune (non-A.I.)disease
controls i p < 0.0002).Responses t o iminunoglobulins from autoimmune and nonautoinimune controls were not signijbantb d$ferent ( p > 0.23).
+
Discussion
Voltage-gated calcium currents measured in the hybrid
motoneuron cell line VSC4.1 appear to represent a
macroscopically homogeneous Ca2+ current population, both electrophysiologically (Mosier DR, Baldelli
P, Delbono 0, et al, manuscript in preparation) and
pharmacologically (defined by lack of current fractions
blocked by inhibitors of L-, N-, and P-type Ca2+ channels as well as the w-CgTx MVIIC-sensitive Q-type
Ca’+ channels [28, 301). These currents, measured using 40 mM Ba7+ as the charge carrier, are completely
blocked by a synthetic analogue of the polyamine funnel-web spider toxin, FTX, in the low micromolar concentration range. However, their relatively low-voltage
threshold of activation, rapid inactivation, and insensitivity to the peptide funnel-web spider toxin, w-AgaIVa, serve to distinguish them from the P-type Ca2+
channels described in a variety of central neurons [26,
27). Presynaptic Car+ currents and action potentialevoked transmitter release at mammalian neuromuscular junctions have been shown to be abolished by FTX
[25}, suggesting that spinal motoneurons and VSC4.1
motoneuron hybrid cells may express a similar class of
Ca’+ channels.
Mosier et
+
al: ALS IgG
Increase Motoneuron Ca” Currents
107
subunit of purified Ca” channels [32). Although
studies of prolonged exposure of neurons to LEMS
IgG have suggested Ca2+ channel down-regulation
[33), the possibility of an early enhancing effect of
LEMS IgG on Caz+ currents will require further investigation.
When comparing these findings with other reports
of ALS immunoglobulin effects on Caz+ channels, we
note that immunoglobulins from 3 ALS patients used
in this study (Patients 1, 2, and 3), which significantly
increased Ba’+ currents in VSC4.1 cells, have been
previously shown to inhibit DHP-sensitive calcium
currents in skeletal muscle fibers [ 111. Immunoglobulin fractions from 2 of these ALS patients (Patients 2
and 3) were recently reported to increase P-type Ca”
currents in cerebellar Purkinje cells and in purified P
channels incorporated into lipid bilayers [ 12). Moreover, immunoglobulins from 3 of the ALS patients
used in this study (Patients 1, 2, and 4 ) increased oCgTx GVIA-sensitive Ba” currents evoked in Xenopus oocytes injected with rat brain messenger R N A
(Delbono 0, Mayer P, Mosier DR, et al, unpublished
data). These actions of ALS immunoglobulins on at
least four different Caz+channel types suggest the possibility that ALS immunoglobulins may bind to epitopes shared between these different CaL+ channels
(possibly located on the a, subunit C32)) or may indirectly modulate their associated regulatory elements.
An alternative possibility is that different immunoglobulins in ALS sera specifically alter the function of individual Ca” channel types. Testing of these different
hypotheses is currently in progress.
While the present data d o not directly address the
ability of ALS immunoglobulins to cause neuronal injury, they do suggest a means by which ALS immunoglobulins can increase intracellular Ca’+, which was
suggested by previous studies of transmitter release in
mammalian motor nerve terminals exposed to ALS
IgG [lo, 341. ALS immunoglobulins can interact directly with voltage-gated Ca2+ channels, as evidenced
by binding studies C32) and by adsorption of Ca2+dependent cytotoxicity of ALS IgG with a purified a1
subunit [l5]. The enhancement of FTX-sensitive Ca2+
currents observed in this study could result from such
a direct interaction, or possibly from an indirect effect
via regulatory elements associated with voltage-gated
Ca2 channels (e.g., kinases, G proteins [35}). This
increase in ETX-sensitive Ca” entry could then lead
to increases in intracellular Ca2+, with a subsequent
contribution to neuronal injury, and is presently under
investigation as a possible first step in the cascade of
events leading to motoneuron death in ALS.
Elevations in intracellular Ca’+ by way of voltagegated Ca2+ channels may also modulate other mechanisms of neuronal compromise proposed to play a role
in ALS, such as excitatory amino acid-mediated toxicOL,
+
108 Annals of Neurology Vol 37
NO 1 January 1995
ity 1361, oxidative injury [2, 31, or alteration of trophic
factor support C371. Alteration of Ca2+ channel function by immunoglobulins in ALS may represent a specific example of a more widely occurring process, with
implications for other disease states, as suggested by
the recent finding that immunoglobulin fracrions from
recent-onset insulin-dependent diabetic patients can increase Ca2+ currents and intracellular Ca2+ in pancreatic p cells [38].
We acknowledge financial support from the NIH, the Muscular Dvstrophy Association, the American Heart Association, and Cephalon,
Inc.
We thank Dr R. Llinas for a generous gift of natural and synthetic
n X . W e thank Dr H. Lopez for provision of neonatal rat D R G
neurons, K. Henry for assistance with culture of VSC4.1 cells, and
M . 3 . Lu for immunoglobulin preparation. Most of all, we are grateful for our patients’ participation in this study and for the inspiration
they have provided in this work.
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