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The Journal of Neuroscience, April 15, 1998, 18(8):2871-2880
G s-Induced Neurodegeneration in
Caenorhabditis elegans
Allison J.
Berger,
Anne C.
Hart, and
Joshua M.
Kaplan
Department of Genetics, Harvard Medical School, Department of
Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts 02114
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ABSTRACT |
We describe a genetic model for neurodegeneration in the nematode
Caenorhabditis elegans. Constitutive activation of the
GTP-binding protein G s induces neurodegeneration. Neuron
loss occurs in two phases whereby affected cells undergo a swelling
response in young larvae and subsequently die sometime during larval
development. Different neural cell types vary greatly in their
susceptibility to Gs-induced cytotoxicity, ranging from
0 to 88% of cells affected. Mutations that prevent programmed cell
death do not prevent Gs-induced killing, suggesting that
these deaths do not occur by apoptosis. Mutations in three genes
protect against Gs-induced cell deaths. The
acy-1 gene is absolutely required for neurodegeneration,
and the predicted ACY-1 protein is highly similar (40% identical) to
mammalian adenylyl cyclases. Thus, Gs-induced
neurodegeneration is mediated by the second messenger cAMP. Mutations
in the unc-36 and eat-4 genes are
partially neuroprotective, which indicates that endogenous signaling
modulates the severity of the neurotoxic effects of Gs.
These experiments define an intracellular signaling cascade that
triggers a necrotic form of neurodegeneration.
Key words:
cell death; neurodegeneration; necrosis; signal
transduction; G-protein; cAMP; mutant; Caenorhabditis
elegans
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INTRODUCTION |
Neuronal cell death is a prominent
feature both of normal brain development and of particular pathological
states. Neuron cell deaths can be placed into two general categories
(apoptotic and necrotic) on the basis of a variety of criteria.
Although a great deal is known about the molecular pathways leading to apoptotic deaths, the pathways leading to necrotic cell deaths are less
well understood. Most examples of necrotic neurodegenerative deaths
occur in pathological states, e.g., stroke (or other cerebrovascular injury) or neurological disorders. One hallmark of these
neurodegenerative disorders is that in each case specific classes of
neurons are targeted for degeneration. For example, dopaminergic
neurons of the substantia nigra are lost in Parkinson's disease,
whereas in epilepsy and focal ischemia CA1 and CA3 neurons of the
hippocampus are killed selectively (Pulsinelli et al., 1982 ; Ben-Ari,
1985).
Genes involved in inherited neurodegenerative disorders have been
characterized in humans as well as in a several model organisms. Nine
genes involved in inherited human neurodegenerative disorders have been
cloned, including huntingtin, ataxin, and SOD. Genetic models for
neurodegeneration also have been described in mice (Mullen et al.,
1976; Herrup and Wilczynski, 1982; Norman et al., 1995), flies (Grether
et al., 1995; Hay et al., 1995; Rangnathanan et al., 1995; Chen et al.,
1996; White et al., 1996), and Caenorhabditis elegans
(Driscoll and Kaplan, 1997; Hengartner, 1997). From these model systems
several genes have been identified that apparently control
neurodegenerative cell deaths. For example, four genes leading to
necrotic forms of neuron death in worms have been described, each
encoding proteins that are similar to mammalian ion channel subunits the epithelial sodium channel homologs (MEC-4, MEC-10, and
DEG-1) and a neuronal acetylcholine receptor homolog (DEG-3) (Chalfie
and Wolinsky, 1990; Driscoll and Chalfie, 1991; Huang and Chalfie,
1994; Treinin and Chalfie, 1995). In these cases neuron death is
thought to occur by exaggerated or toxic influx of ions, perhaps akin
to excitotoxicity in mammals.
Although these genetic studies successfully have identified the many
genes involved in neurodegenerative deaths, in most cases the identity
of these genes has not implicated directly a defined signal
transduction pathway in neurodegeneration. Alterations in
G-protein-coupled phospholipase C signaling lead to retinal degeneration in Drosophila (for review, see Rangnathanan et
al., 1995). A second example of G-protein-induced neurodegeneration has
been reported recently. Constitutive activation of the heterotrimeric G-protein Gs causes neuronal degeneration in C. elegans (Korswagen et al., 1997). Here we show that cells differ
greatly in their susceptibility to Gs-induced killing,
that the neurotoxic effects of Gs are mediated by
acy-1 (which encodes a protein that is 40% identical to
mammalian adenylyl cyclases), and that endogenous neural signaling
modulates the severity of Gs-induced killing. These
results define an intracellular signaling pathway by which Gs triggers a necrotic form of neuron death.
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MATERIALS AND METHODS |
Plasmid construction and transgene expression. The
Gs expression vector (KP#20) was constructed by ligating
a 1.5 kb NcoI-XhoI fragment encoding a GTPase
defective (Q227L) mutant rat Gs cDNA containing the
hemagglutinin (HA) epitope into the glr-1 expression vector
CX#1 (Hart et al., 1995; Maricq et al., 1995). A
mec-7:: s(gf) expression
plasmid (KP#7) was constructed by ligating the 1.5 kb
NcoI-XhoI Gs(Q227L) into the
mec-7 expression vector pPD52.102. Transgenic animals were
prepared by microinjecting various expression constructs together with
a glr-1:: gfp plasmid (KP#6), using
lin-15 as a transformation marker (Huang et al., 1994). Two
stable lines carrying glr-1 expression constructs for both
green fluorescent protein (GFP) and the GTPase defective
Gs (nuIs3 and nuIs5) were isolated
after  -irradiation. Unless otherwise noted, data reported in the
text refer to nuIs5, which is referred to as
s(gf). Integrated lines typically
greatly overexpress the transgenes. Thus, it is likely that the
nuIs5-encoded Gs is more abundant than the
endogenous C. elegans Gs.
Characterization of the neurodegeneration phenotype. Swollen
or missing cells were identified by examining the morphology of
GFP-expressing cells. The glr-1-expressing cells were
identified previously (Hart et al., 1995; Maricq et al., 1995). In
s(gf) animals ~90% of the PVC
neurons degenerate. Other cells degenerate at lower frequencies,
including AVA, AVD, AVE, AVG, PVQ, RIG, and SMD. Cell deaths with
similar morphology and similar cell type specificity were observed in
both nuIs3 and nuIs5 animals. Gs-induced neurodegeneration in various genetic
backgrounds was quantitated as the number of swollen PVC neurons in L1
larvae and as the percentage of PVC neurons that are missing or are
swollen in adult hermaphrodites. Statistical differences between
genotypes was determined by the method of attributable risk (Devore,
1987). We compensated for multiple comparisons by setting
p < 0.005 as the threshold for significance.
Confidence intervals of 95% were calculated as 1.96* (SEM).
Antibody staining. Gs expression in
transgenic animals was monitored by staining fixed animals with anti-HA
antibodies. Fixation and antibody staining were done according to a
protocol devised by M. Nonet. Briefly, worms were fixed and their
cuticles reduced in Bouin's solution with  -mercaptoethanol (BME).
Worms were washed sequentially in BTB [1× borate buffer, pH 9.2 (20 mM H3BO3 and 10 mM
NaOH), 0.5% Triton X-100, and 2% BME], BT (1× borate buffer and
0.5% Triton X-100), and finally in AbA solution (1× PBS, 1% BSA,
0.5% Triton X-100, 0.05% sodium azide, and 1 mM EDTA).
The anti-HA monoclonal antibody 12CA5 (Boehringer Mannheim,
Indianapolis, IN) and goat anti-mouse rhodamine-conjugated antibody
(Cappel, Cochranville, PA) were used at a 1:100 dilution, and
incubations were done in AbA solution overnight at room
temperature.
Isolation of acy-1 mutations. Mutations that
restore normal locomotion rates to
s(gf) homozygotes were isolated
from the F2 self-progeny of EMS mutagenized hermaphrodites. Candidate
suppressor mutants subsequently were screened for the reduction of
Gs-induced swelling in L1 larvae. In a screen of 7500 haploid genomes, the nu327, nu329, and
nu343 alleles were isolated. Other suppressor mutations
isolated in this screen will be described elsewhere.
Positional cloning of acy-1. The acy-1 mutations
nu327, nu329, and nu343 are all linked
to dpy-17 in two-factor mapping experiments. Three-factor
mapping placed acy-1 between emb-5 and
dpy-17: (nu327 dpy-17) 37/37
unc-32; (nu329 dpy-17) 16/16
unc-32; (nu343 dpy-17) 4/4
unc-32; unc-79 (6/14) MJ#NEC2 (5/14)
nu329 (3/14) dpy-17; emb-5 (1/16)
nu327 (15/16) dpy-17. The cosmid F17C8 (which
carries ACY-1) was microinjected into acy-1(nu327); nuIs5
animals, and transgenic lines were isolated by using
goa-1:: gfp (KP#13) as a transformation marker
(Ségalat et al., 1995). Four independent lines were obtained, two
of which were rescued for the acy-1 phenotype, i.e., they
had increased degeneration of the PVC neurons. Sequences spanning the
GENEFINDER-predicted exons of ACY-1 (accession number Z35719) were
amplified from the mutants nu327, nu329, and
nu343, and the resulting fragments were sequenced directly
by cycle sequencing. The GENEFINDER prediction for the first exon was
confirmed by isolating partial cDNA clones from the Barstead RB1 cDNA
library by PCR amplification.
Analysis of acy-1 expression. A deleted
derivative (KP#106) of the cosmid F17C8 was isolated by digestion with
AflII and religation. KP#106 contains the entire 8.35 kb of
the acy-1 genomic region, together with 5.2 kb 5' and 4.9 kb
3' flanking sequences. An acy-1:: gfp expression
vector (KP#107) was constructed by PCR-amplifying a 1.7 kb fragment
containing the GFP coding region and the unc-54 transcription terminator from pPD95.75 and ligating this fragment into
the unique Asp718 site in KP#106, creating a fusion protein containing the first six exons of acy-1 fused to GFP. The
ACY-1:: GFP fusion protein contains six predicted
transmembrane domains of ACY-1 and hence is membrane-localized.
Transgenic animals carrying KP#107 were isolated by microinjection,
using lin-15 as a transformation marker (Huang et al.,
1994). Expressing cells were identified on the basis of their
morphology and nuclear positions.
Isolation and responsiveness of eat-4 mutations.
We screened 11,000 mutagenized haploid genomes for animals that
failed to respond to nose touch. Mutants isolated were subjected to a
series of secondary screens, including dye filling of the amphid
sensory neurons and responsiveness to osmotic shock and volatile
repellents. Six alleles of eat-4 (n2458,
n2474, nu2, nu142, nu143,
and nu146) were isolated in this screen. A seventh
allele, eat-4(ky5), was isolated by C. Bargmann (University
of California at San Francisco, San Francisco, CA) as a chemotaxis
defective mutant. All seven eat-4 alleles are normal for dye
filling but are defective for all three ASH sensory behaviors.
Behavioral assays. ASH sensory responses were assayed as
previously described (Kaplan and Horvitz, 1993; Hart et al., 1995; Maricq et al., 1995; Troemel et al., 1995). For nose touch, animals were tested 10 times each, with a positive response being scored when
animals either halted forward movement or initiated backward movement
after the stimulus. Osmotic avoidance assays were performed with either
of two protocols. Assays in Table 3 were done as described previously
(Hart et al., 1995). Assays in Table 2 were done with a modified
protocol described by C. de Vries and R. Plasterk (personal
communication). Briefly, worms were washed twice with S-basal and once
with water and placed inside a semicircle of 60% glycerol with
bromphenol blue. A microliter of diacetyl at a 1:100 dilution was
placed outside the semicircle at the opposite edge of the plate. The
percentage of adult worms that crossed the osmotic barrier after 10 min
was calculated. For ASH-mediated volatile avoidance, an eyelash was
dipped in 1-octanol and held near an animal's nose; responses were
quantitated by recording the length of time that elapsed before an
animal reversed locomotion.
Chemotaxis assays were performed as previously described (Bargmann et
al., 1990). Assay plates contained 2% Difco-agar, 5 mM
potassium phosphate, pH 6.0, 1 mM calcium chloride, and 1 mM magnesium sulfate. Animals were placed at the center of
the plate, with 1 ml of diluted attractant and 1 ml of 1 M
sodium azide at one edge of the plate; 1 ml of ethanol and 1 ml of
sodium azide were placed at the opposite edge. Dilutions in ethanol
were as follows: 1:1000 for diacetyl, 1:100 for isoamyl alcohol, and
1:200 for benzaldehyde. The chemotaxis index was calculated after 1 hr
as the (number of worms at attractant  number of worms at control solvent)/total number of animals.
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RESULTS |
Gs-induced neurodegeneration
While studying signaling by the G-protein Gs,
we made the observation that expression of a constitutively active rat
Gs cDNA caused neurodegeneration in C. elegans. Mutations that diminish the GTPase activity of
Gs have been shown to cause constitutive, agonist-independent signaling (Landis et al., 1989; Lyons et al., 1990;
Wong et al., 1991). We expressed a rat cDNA encoding a GTPase-defective (Q227L) Gs subunit, hereafter referred to as
s(gf), in C. elegans neurons by using the glr-1 glutamate receptor (GluR)
promoter. We chose the glr-1 promoter because
glutamate-responsive cells might be prone to neurodegeneration, because
it is highly expressed, and because glr-1-expressing cells
control locomotion, an easily assayed behavior. The glr-1
promoter is expressed in 17 classes of neurons, including interneurons
required for locomotion (Hart et al., 1995; Maricq et al., 1995).
Gs was coexpressed with the GFP protein of
Aequorea victoria (Chalfie et al., 1994), which allowed us
to examine the morphology of Gs-expressing cells. Transgenic glr-1:: s(gf)
animals were paralyzed and a subset of the expressing neurons swelled
to several times their normal diameter and eventually disappeared,
presumably because they died (Fig. 1,
Table 1). These results suggest that
exaggerated Gs signaling kills neurons.
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Figure 1.
Neurodegenerations caused by activated
Gs. A GTPase-defective rat Gs cDNA
containing the HA epitope tag was coexpressed with the green
fluorescent protein (GFP) in GLR-1-expressing neurons, as described in
Materials and Methods. Neurodegeneration occurs in two phases. In young
larvae (A, B) affected cells swell to
several times their normal diameter. Swelling is apparent by the
morphology of GFP-expressing cells (A) and by
their appearance in bright-field optics (B) as
enlarged and apparently vacuolated cells, often with an intact nucleus.
The interneurons AVE and AVD are swollen, compared with neighboring
unaffected cells (asterisks). Cytotoxicity is scored by
examining adult animals for the presence of the PVC neuron.
GFP-expressing PVC neurons typically are missing in
s(gf)
(C), but not in
s(gf); acy-1(nu343)
(D) adults. Expression of Gs was
monitored in s(gf);
acy-1(nu343) animals by staining with anti-HA antibodies
(E). This panel shows a stained L1 larva.
Although some differences in expression levels are apparent, typically
10 brightly staining neurons are seen in the lateral ganglion. Staining
of a PVC neuron is shown for comparison.
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Gs-expressing neurons differed greatly in their
susceptibility to Gs-induced toxicity. In first-stage
(L1) glr-1:: s(gf) larvae, the swelling of different cell types occurred at very different
frequencies, and these differences were seen in two independent
s(gf) transgenes: in
nuIs5, PVC 88%, AVD 34%, and RIG 7%; in nuIs3,
PVC 77%, AVD 44%, and RIG 6%. One potential mechanism for this
apparent cell type specificity would be that cells differ substantially
in their levels of Gs expression. Because the rat
Gs cDNA contains the HA epitope, we were able to test
this possibility by staining s(gf)
animals with anti-HA antibodies (Fig. 1E).
Differences in Gs expression correlated well with
differences in toxicity for some cells, but not for others. For
example, RIG neurons expressed much less Gs and swelled much less frequently than PVC neurons, whereas AVD and PVC neurons expressed equivalent amounts of Gs but swelled at
significantly different frequencies. In general, 10 neurons in the
lateral ganglion expressed levels of Gs similar to those
seen in the PVC, whereas most
s(gf) animals have only one or two
dying cells in the lateral ganglion (Fig. 1A). Thus,
many more cells express Gs than are found to die, and
differences in Gs-induced toxicity do not always correlate with differences in Gs expression. To
demonstrate further the specificity of Gs-induced
neurodegeneration, we expressed s(gf) by using the
mec-7 promoter. MEC-7 tubulin is expressed abundantly in
five neurons that sense light touch to the worm's body, which are
called touch cells (Savage et al., 1989; Hamelin et al., 1992; Mitani
et al., 1993). Animals expressing the
mec-7:: s(gf) transgene
are indistinguishable from wild-type animals, having no obvious defect
in touch sensitivity nor in the morphology of the touch cells (data not
shown). Therefore, both the glr-1 and the mec-7
expression constructs support the notion that the effects of
Gs on neural activity and on neurodegeneration are cell
type-specific.
The pattern of cell deaths that was observed does not explain the
severity of the locomotion defect in
s(gf) animals. The GLR-1-expressing interneurons AVA, AVB, AVD, and PVC play an important role in locomotion; hence these cell deaths could, in principle, explain the locomotion defects (Chalfie et al., 1985). However, the
sluggish locomotion defect is apparent in 100% of the
s(gf) animals, although cell
deaths are found in only a subset of animals (see above). For example,
a significant fraction of uncoordinated animals can be found in which
only the PVC neurons have died (A. Berger and J. Kaplan, unpublished
observations). Because killing the PVC neurons with a laser microbeam
is not sufficient to cause uncoordinated locomotion (Chalfie et al.,
1985), these results suggest that the
s(gf) transgene inhibits the
function of these interneurons in addition to causing a subset of these
cells to die.
ACY-1 mediates Gs-induced neurodegeneration
To identify the targets of Gs, we isolated
mutations that block Gs-induced paralysis and
neurodegeneration. In a screen of 7500 haploid genomes, we isolated
three semidominant mutations that map to the cluster of chromosome III
(Fig. 2, Table 1). Via a series of
experiments we showed that these mutations occur in an adenylyl cyclase
gene, which we have named acy-1. First, two of these
mutations were mapped to a 1.5 cm genetic interval between MJ#NEC2 and
dpy-17 (Fig. 2A). Second, a transgene
carrying a cosmid from this interval (F17C8) corrected the mutant
phenotype of acy-1(nu327) animals (Fig.
2B). Third, all three alleles corresponded to
mutations in the predicted exons of the gene F17C8.1 (Fig. 2C,D), one of two predicted adenylyl cyclase genes in the
C. elegans genome database. It is unclear why these
mutations are partially dominant. The molecular nature of the
mutations, together with the fact that the mutant phenotype is rescued
by a wild-type copy of the F17C8 cosmid, suggests that these mutations
reduce ACY-1 activity. For example, nu329 and
nu343 are predicted to disrupt pre-mRNA splicing.
Thus, it is possible that s(gf)
animals are highly sensitive to changes in cAMP levels; however,
because none of the genetic deficiencies in this region uncovers the
acy-1 gene, we cannot test directly whether acy-1
is haplo-insufficient. These results suggest that Gs
neurodegeneration is mediated by changes in intracellular cAMP.
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Figure 2.
ACY-1 encodes an adenylyl cyclase.
A, Genetic and physical map position of
acy-1. B, Transgenes containing the
cosmid F17C8 rescue the acy-1(nu327) mutant phenotype.
C, The predicted amino acid sequence, as predicted by
GENEFINDER (accession number Z35719Z35719) and confirmed by our RT-PCR
analysis, of ACY-1 (top) is shown aligned to mouse
adenylyl cyclase type 9 (bottom), the most highly
related sequence (40% identical) in the database.
Underlined sequences indicate predicted transmembrane
domains. D, Predicted structure of the
acy-1 gene (using the GENEFINDER algorithm) and of the
GFP fusion construct (KP#107) are shown. Positions of the
acy-1 mutations nu327,
nu343, and nu329 are indicated
(C, D).
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The expression pattern of acy-1 was determined by analyzing
a GFP reporter construct. The acy-1:: gfp fusion
protein is expressed in virtually all neurons and body muscles;
however, it does not appear to be expressed in other tissues (Fig.
3). Therefore, the cell type specificity
of Gs-induced neurodegeneration cannot be explained by
the expression pattern of the acy-1:: gfp reporter. One caveat to this conclusion is that the expression of reporter genes
can differ from that of the endogenous genes if, for example, some
critical regulatory elements are missing in the reporter constructs.
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Figure 3.
ACY-1 is expressed in neurons and muscles. The
KP#107 acy-1:: gfp fusion gene was expressed in
transgenic animals, as described in Materials and Methods. ACY-1
appears to be expressed in most or all muscles and neurons.
A, Expression in the two ventral rows of body muscles
(arrows) and in the ventral cord neurons and neuropile
(lines). B, Expression in the vulva
muscles (arrowheads). Nearly all of the 302 neurons in
the adult appear to express ACY-1. Cell bodies can be identified on the
basis of the bright fluorescence in intracellular membranes (presumably
the endoplasmic reticulum or Golgi apparatus). ACY-1 does not appear to
be expressed in non-neural tissues, nor is it expressed in the
pharynx.
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Role of ACY-1 in sensory behaviors
cAMP has been implicated in a wide variety of signaling pathways,
and ACY-1 is expressed in most if not all neurons and muscles in the
worm. Therefore, we would expect that acy-1 mutants would have defects in behavior or development. In particular, the C. elegans Gs subunit was shown previously to be
essential for viability as well as for regulating locomotion and egg
laying (Korswagen et al., 1997); hence we would expect to find similar
defects in acy-1 mutants. Surprisingly, acy-1
mutants are overtly normal, with no obvious defects in development,
fertility, egg laying, or male mating behaviors (data not shown).
Because the deaths of GLR-1-expressing cells are prevented by
acy-1 mutations, we tested whether other behaviors mediated
by GLR-1-expressing cells are impaired by acy-1 mutations.
GLR-1-expressing cells are required for response to body touch and for
locomotion (Chalfie et al., 1985); however, these behaviors are not
affected in acy-1 mutants (see Table 4) (our unpublished
observations). Three sensory behaviors mediated by the ASH neurons
(nose touch, osmotic shock, and volatile repellent responses) are also
likely to be mediated by the GLR-1-expressing cells (Bargmann et al.,
1990; Hart et al., 1995; Maricq et al., 1995; Troemel et al., 1995),
yet none of these behaviors is impaired in acy-1 mutants
(Table 2).
Recently, two putative cyclic nucleotide-gated ion channels (TAX-2 and
TAX-4) were shown to be involved in axon morphogenesis, thermotaxis,
and olfaction (Coburn and Bargmann, 1996; Komatsu et al., 1996).
Because these channels in principle could be activated by the cAMP
produced by ACY-1, we examined acy-1 mutants for defects in
these processes (Table 2). Mutations in tax-2 and
tax-4 cause certain chemosensory neurons to grow out
supernumerary axonal processes, which can be visualized by staining the
animals with the fluorescent dye DiI (Coburn and Bargmann, 1996;
Komatsu et al., 1996). We found no abnormal sensory axon morphologies
in DiI-stained acy-1 mutants (data not shown). Chemotaxis
toward isoamyl alcohol and benzaldehyde requires TAX-2 and TAX-4
channels (Coburn and Bargmann, 1996; Komatsu et al., 1996). We found
that the response of acy-1 mutants to benzaldehyde was
normal, whereas their response to isoamyl alcohol was impaired
slightly. Finally, the thermotactic behavior of acy-1
mutants was also normal (A. Berger and O. Hobert, unpublished
observations). Because acy-1 mutants lack the axon and
olfactory defects seen in tax-2 and tax-4
mutants, it is unlikely that ACY-1 is the sole source of cyclic
nucleotides required for the activation of TAX-2 and TAX-4. These
results are consistent with the fact that TAX-4 channels are activated
selectively by cGMP (Komatsu et al., 1996). Furthermore, these results
do not exclude the possibility that cAMP is required for these
behaviors, because there is at least one other adenylyl cyclase in the
worm genome.
Cytotoxic targets of cAMP
Several previously identified genes were considered good
candidates for mediating the toxic effects of Gs (see
Table 1). The putative cyclic nucleotide-gated ion channels TAX-2 and
TAX-4 are not expressed in glr-1-expressing cells (Coburn
and Bargmann, 1996; Komatsu et al., 1996) and hence are unlikely
targets in this case. The mec-6, unc-8, and
deg-1 genes were implicated previously in neurodegeneration
(Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991; Shreffler et
al., 1995; Tavernarakis et al., 1997), and the DEG-1 and UNC-8 proteins
are similar to mammalian epithelial sodium channel subunits (ENaC),
which are activated potently by cAMP-dependent protein kinase (PKA)
(Sariban-Sohraby et al., 1988; Oh et al., 1993; Bubien et al., 1994).
The unc-2, unc-36, and egl-19 genes
encode subunits of voltage-dependent Ca2+ channels
(Schafer and Kenyon, 1995; Lee et al., 1997) that are likely to be
regulated by PKA (Curtis and Catterall, 1985) and also have been
implicated in neurodegeneration. The glr-1 gene encodes an
ionotropic GluR (Hart et al., 1995; Maricq et al., 1995), GluRs have
been implicated in neurodegeneration in mammals (Olney, 1986; Choi,
1988), and PKA augments the response of mammalian neurons to
glutamatergic agonists (Greengard et al., 1991; Wang et al., 1991;
Colwell and Levine, 1995).
Of these candidate genes the unc-36 mutations
(e251 and e837) conferred a slight but
significant reduction in Gs-induced cytotoxicity (Table
1). Interestingly, the unc-36 mutations had no effect on
cell swelling. These results suggest that either Ca2+ influx or depolarization of the affected cells
modulates susceptibility to Gs cytotoxicity. All other
candidate genes had no effect on either neuron swelling or deaths in
glr-1:: s(gf) animals
(Table 1). Our results do not exclude the possibility that these other candidate PKA targets play a role in Gs-induced
toxicity. For example, more than one type of channel may be capable of
mediating the toxic effects of Gs, in which case
neurodegeneration would be prevented only in multiply mutant
animals.
Role of apoptosis and necrosis in
Gs neurodegeneration
Whether neurodegeneration occurs by apoptosis or by necrosis has
remained controversial (Choi, 1996). The Gs-induced
deaths appear to be necrotic (i.e., undergoing cell swelling and
delayed cytotoxicity) rather than apoptotic. We directly tested the
role of apoptosis in these deaths by testing the effects of mutations in cell death genes (Hengartner, 1997). We found that mutations in the
cell death genes ced-3 and ced-4, both of which
are required for apoptosis (Ellis and Horvitz, 1986), had no effect on
Gs-induced swelling or killing (Table 1). Thus,
apoptosis is not required for Gs-induced killing.
Role of endogenous neural signaling in
Gs-induced neurodegeneration
Because Gs often couples to neurotransmitter
receptors thereby producing or altering synaptic transmission, we
wondered whether endogenous neural activity would regulate
Gs-induced neurodegeneration. The weakly neuroprotective
effect of unc-36 mutations is consistent with this
hypothesis. Decreased calcium influx or decreased cell excitability in
unc-36 mutants could explain this neuroprotective effect. In
addition, unc-36 mutations have been shown to decrease endogenous synaptic transmission, albeit by an uncharacterized mechanism (Nguyen et al., 1995). Because UNC-36 channels play a role in
many different aspects of neural activity, we reasoned that mutations
that more specifically perturb the cytotoxic mechanism might produce a
more profound neuroprotective effect.
To test directly the role of synaptic activity on these cell deaths, we
tested the effect of mutations in the unc-104 and snt-1 genes, which encode phylogenetically conserved
proteins (kinesin heavy chain and synaptotagmin) that are required for synaptic vesicle transport and exocytosis (Otsuka et al., 1991; Nonet
et al., 1993). We found that neither the unc-104 nor
snt-1 mutations reduced Gs-induced cell
killing. This result suggests that the overall levels of synaptic input
are not required for killing, per se.
Given its role in excitotoxicity in mammals, we wondered whether
endogenous glutamate signaling is required for Gs
neurodegeneration. We found that a loss-of-function glr-1
mutation did not reduce Gs-induced cell swelling or
cytotoxicity (Table 1). This result does not exclude the possibility
that glutamate neurotransmission mediates cAMP-induced cytotoxicity.
The C. elegans genome sequence (currently ~85% complete)
predicts eight additional ionotropic GluR subunits; therefore, the
glr-1 mutation is unlikely to eliminate glutamate signaling
in vivo.
eat-4 mutations are neuroprotective
Because GLR-1 receptors are required for several mechanosensory
behaviors (Hart et al., 1995; Maricq et al., 1995), we reasoned that
other mutations affecting these behaviors might be neuroprotective. Previous work has shown that ASH sensory neurons mediate an aversive response to three distinct stimuli (nose touch, osmotic shock, and
volatile repellents) and that the ASH-mediated touch response requires
functional GLR-1 glutamate receptors in synaptic targets of ASH (Hart
et al., 1995; Maricq et al., 1995; Troemel et al., 1995). We isolated
seven alleles of the eat-4 gene, originally identified
because of its function in pharyngeal pumping (Avery, 1993), in a
screen for mutations that eliminate ASH-mediated touch sensitivity. All
seven eat-4 strains have similar behavioral defects. In
particular, they have severe defects in the ASH-mediated touch, osmosensory, and volatile repellent responses (Table
3). We found that eat-4
mutations significantly reduced Gs-induced cytotoxicity but had no apparent effect on cell swelling (see Table 1). PVC cytotoxicity in eat-4 unc-36;
s(gf) triple mutants (55 ± 5%) was not significantly different from that seen in unc-36;
s(gf) double mutants, suggesting that
eat-4 and unc-36 act in a single pathway
regulating Gs-induced killing. In addition,
eat-4 mutations dramatically improved the locomotion rate of
s(gf) animals (Table 4). Thus, the eat-4 gene plays
a significant role in Gs-induced effects on
neurodegeneration (reducing cytotoxicity) and on neural activity
(reducing paralysis).
| |
DISCUSSION |
We and others (Korswagen et al., 1997) have shown that the
expression of a constitutively active form of Gs induces
a form of neurodegeneration in the nematode C. elegans. The
Gs-induced deaths appear to be necrotic, because the
affected cells swell and subsequently lyse and because these deaths are
not prevented by mutations in the cell death genes ced-3 and
ced-4. We provide here a detailed analysis of the
Gs killing pathway. Neurons differ greatly in their
susceptibility to Gs-induced neurodegeneration, ranging
from 0 to 88% killed. Three genes (acy-1, eat-4,
and unc-36) that contribute to
Gs-induced neurodegeneration are identified. Gs killing appears to be mediated by the second
messenger cAMP, because acy-1 mutations block killing. Our
data also suggest that the two phases of Gs
neurodegeneration can be distinguished genetically, because
acy-1 mutations block both swelling and cytotoxicity, whereas other mutations (i.e., unc-36 and
eat-4) reduce cytotoxicity but have no effect on
swelling. However, because we have not quantitated the extent or
duration of cell swelling, it remains possible that there is a more
subtle correlation between the extent of swelling and cytotoxicity.
Finally, killing is not dependent on synaptic transmission, but it is
modulated by endogenous neural signaling.
Function of ACY-1
We have isolated mutants that lack ACY-1, an adenylyl cyclase.
ACY-1 appears to be expressed in nearly all neurons and muscles, but
not in other tissues. These results suggest that ACY-1 adenylyl cyclase
is likely to participate in many neural signaling pathways. Therefore,
we would expect that acy-1 mutants would have defects in
behavior or development. Consistent with this notion is that mutations
that inactivate the C. elegans Gs subunit
(GSA-1) are homozygous lethal (Korswagen et al., 1997). Surprisingly,
the behavior and morphology of acy-1 homozygotes are nearly
indistinguishable from wild-type animals. Several behaviors were
examined in greater detail, including behaviors mediated by
GLR-1-expressing cells, behaviors regulated by GSA-1, and those that
require the cyclic nucleotide-gated channels TAX-2 and TAX-4. We found
that acy-1 mutants were proficient in all of these
behaviors. These results suggest that acy-1 alleles do not
eliminate ACY-1 activity, that the function of GSA-1 required for
viability and for regulating behaviors is mediated by another adenylyl
cyclase, or that ACY-1 acts redundantly with other forms of adenylyl
cyclase. The genome sequence predicts at least one other adenylyl
cyclase gene, which could account for the discrepancy between the
gsa-1 and acy-1 mutant phenotypes. Our results
also suggest that ACY-1 is not the sole source of cyclic nucleotides
required for the activation of TAX-2 and TAX-4, which is consistent
with previous studies suggesting that TAX-4 channels are activated
selectively by cGMP.
Role of cAMP in neurodegeneration
Several possible mechanisms could explain the toxic effects of
activated Gs. We believe that the most likely
explanation is that cAMP regulation of ion channels or ion transporters
grossly alters membrane permeability, leading to cell swelling and
death. In other systems many ion channels have been shown to be
potently regulated by cAMP. We tested the C. elegans
homologs of several of these potential cAMP targets, finding that
mutations reducing the activity of UNC-36 calcium channels had a modest
but significant neuroprotective effect. Because voltage-dependent
calcium channels correspond to heteromultimers of several types of
subunits, it is difficult to predict the extent to which alteration of
the UNC-36 2 subunit reduces the overall calcium permeability of cells in vivo. Although other candidate mutations were not
neuroprotective, these results do not exclude a role for these genes,
e.g., if several genes play redundant roles in inducing
neurodegeneration.
An alternative explanation for Gs-induced
neurodegeneration is that cells expressing the Gs
transgene have taken on the fates of cells that undergo developmentally
programmed cell deaths. Several facts argue against this model. First,
all of the cell deaths that occur during normal C. elegans
development are apoptotic, by both morphological and genetic criteria
(Hengartner, 1997). Therefore, the necrotic cell deaths produced by
Gs are not seen in normal development. Second, the
glr-1 promoter used to express Gs is
expressed starting in the threefold embryo (Hart et al., 1995; Maricq
et al., 1995) after most cell fate choices (and in fact most cell
deaths) already have occurred (Sulston et al., 1983). Third,
Gs-expressing cells continue to express the
glr-1 promoter, indicating that at least some aspects of
cell fate have not been altered.
It is also possible that overproduction of cAMP diminishes ATP levels,
leading to a metabolic crisis and cell death. We think that this model
is unlikely for three reasons. First, Gs-induced cell
deaths are cell type-specific, which would not be predicted by this
model. Second, suppression by acy-1 mutations is
semidominant, which implies that cells are sensitive to subtle changes
in cAMP levels. Third, mutations that alter endogenous neural signaling (i.e., unc-36 and eat-4) modulate
Gs toxicity, implying that the toxic signal is mediated
by normal signaling pathways. These results suggest that, rather than
creating a catastrophic metabolic event, Gs kills via
endogenous signaling pathways. On the other hand, the terminal phases
of cytotoxicity must include the gross alteration of cellular
metabolism and cellular integrity. Thus, in addition to inducing a
cytotoxic signal, overproduction of cAMP also might hasten death by
acting as a metabolic sink.
cAMP has been implicated in growth control and cell death in other
systems. For example, elevated cAMP levels are cytotoxic in S49
lymphoma cells (Coffino et al., 1975), and these deaths subsequently
have been shown to occur by apoptosis (Lanotte et al., 1991; Duprez et
al., 1993). The molecular target of cAMP in the induction of lymphoid
apoptosis has not been determined. In other cell culture systems cAMP
induces growth arrest in G1 of the cell cycle (Khan et al., 1996). To
our knowledge this is the first report of cAMP-induced
neurodegeneration. In fact, in several other models of
neurodegeneration cAMP has been shown to be neuroprotective (D'Mello
et al., 1993; Dockwerth and Johnson, 1993; Kawakami et al., 1996;
Michel and Agid, 1996).
Role of eat-4 and unc-36
We found that eat-4 and unc-36 mutations are
partially protective against the cytotoxic and paralytic effects of the
Gs transgene. Although these protective effects are
admittedly subtle, we believe that they are real for several reasons.
First, all of our data were compared by statistical methods, using a
relatively strict threshold for significance (p < 0.005). Second, we have examined many other genetic backgrounds and
found no similar protective effects, which indicates that effects of
this magnitude are uncommon. Third, for both eat-4 and
unc-36 we found the neuroprotective effect in two unrelated
strains carrying different alleles of these genes. Therefore, it is
highly likely that these protective effects are caused by the mutations
in unc-36 and eat-4 rather than by some
uncharacterized mutation in the genetic background. By all of these
measures the effects of the eat-4 and unc-36
mutations are real; therefore, we conclude that these genes play some
role in determining the severity of Gs-induced
killing.
The eat-4 gene was identified initially in screens for
mutations that disrupt eating behavior (Avery, 1993). The
eat-4 eating defect is caused by the elimination of a
glutamate-induced inhibitory synaptic signal (mediated by the M3 motor
neuron), which can be observed in extracellular recordings of
pharyngeal muscle activity (Raizen and Avery, 1994). The defect in
pharyngeal neurotransmission is likely to be caused by a presynaptic
defect in M3, because pharyngeal muscles isolated from eat-4
mutants are responsive to glutamate iontophoresis (Dent et al., 1997).
We have shown that eat-4 mutants are defective for three
ASH-mediated sensory behaviors, one of which (nose touch) is mediated
by GLR-1 GluRs. Thus, both the eating defects and the sensory defects
observed in eat-4 mutants could be explained by an
underlying defect in glutamate neurotransmission. The eat-4
gene has been cloned (R. Lee, E. Sawin, M. Chalfie, H. R. Horvitz, and
L. Avery, personal communication); however, the molecular identity of
EAT-4 does not reveal what role it plays in neuronal signaling.
Two sorts of models could explain the neuroprotective effects of
eat-4 and unc-36 mutations. First, these
mutations could be neuroprotective, because Gs-induced
deaths are mediated in part by endogenous glutamate neurotransmission.
Alternatively, EAT-4 and UNC-36 could act in the dying cells, directly
or indirectly mediating the cytotoxic effects of cAMP.
Gs-induced killing is not diminished by mutations that
impair synaptic transmission, which would favor the model that EAT-4
and UNC-36 act in the dying cells. However, EAT-4 is not expressed in
PVC neurons (R. Lee, E. Sawin, M. Chalfie, H. R. Horvitz, and L. Avery, personal communication), which favors the idea that EAT-4 acts
in the presynaptic partner. Neither of these results conclusively tests
these models. The reported EAT-4 expression pattern may be incomplete.
Moreover, several results indicate that residual synaptic transmission
occurs in unc-104 and snt-1 mutants. The
unc-104 allele used in this study is a partial loss of
function, because null alleles are homozygous lethal (Hall and
Hedgecock, 1991), and residual synaptic transmission has been
documented in synaptotagmin null mutants in worms, flies, and mice
(DiAntonio et al., 1993; Littleton et al., 1993; Nonet et al., 1993;
Geppert et al., 1994). Moreover, some glutamate may be released by a
nonvesicular mechanism (Attwell et al., 1993). In fact, cell swelling
might stimulate glutamate efflux through volume-sensitive osmolyte
channels (Jackson and Strange, 1993). Finally, it is also possible that
defects in exocytosis make cells more susceptible to necrosis, for
example by preventing the addition of new membranes to swelling cells.
Further experiments will be required to distinguish among these
models.
Similarities to other forms of neurodegeneration
Several other C. elegans mutations have been described
that cause a necrotic form of neurodegeneration (Chalfie and Wolinsky, 1990; Driscoll and Chalfie, 1991; Treinin and Chalfie, 1995). In
particular, mutations in the deg-1 gene also cause a
specific subset of neurons to undergo necrosis, including the PVC
neuron (Chalfie and Wolinsky, 1990; Garcia-Anoveros et al., 1995). Our results suggest that the deg-1-induced and
Gs-induced cell deaths occur by distinct mechanisms
because mec-6 mutations block deg-1-induced deaths, but not Gs-induced cell deaths (Chalfie and
Wolinsky, 1990); however, it remains possible that ACY-1 is required
for deg-1-induced deaths.
Gs-induced cell deaths also share some properties with
glutamate-induced excitotoxicity in mammals. Neurodegeneration occurs in two phases (swelling and killing), and killing is apparently cell
type-specific. Reducing the activity of voltage-dependent calcium
channels is neuroprotective in both cases; however, the protective
effect of unc-36 mutations is modest (albeit statistically significant). Finally, the neuroprotective effects of eat-4
mutations imply that glutamate may play a role in
Gs-induced neurodegeneration. Further evidence will be
required to determine whether Gs-induced cell deaths and
excitotoxicity are related mechanistically.
| |
FOOTNOTES |
Received Dec. 5, 1997; revised Jan. 20, 1998; accepted Jan. 27, 1998.
This work was supported by Grants from National Institutes of Health
(NS32196) and the Human Frontiers Science Program to J.K. A.B. was
a Howard Hughes Medical Institute predoctoral fellow. A.H. was
supported by a fellowship from the Medical Foundation. J.K. is a Pew
Scholar in the Biomedical Sciences. We thank L. Chen for excellent
technical assistance; A. Fire, F. Kolakowski, and H. Bourne for
plasmids; A. Coulson for cosmids; R. Barstead for cDNA libraries; R. Horvitz, M. Chalfie, J. Miwa, E. Jorgensen, and the C.
elegans Genetics Stock Center for strains; M. Nonet for
providing the antibody staining protocol; and C. Bargmann for the
eat-4(ky5) allele.
Correspondence should be addressed to Dr. Kaplan at his present
address: Department of Molecular and Cell Biology, 361 Life Sciences
Addition, University of California, Berkeley, CA 94720-3200.
Dr. Hart's present address: Department of Pathology, Harvard Medical
School and Massachusetts General Hospital Cancer Center, 149-7202 13th
Street, Charlestown, MA 02129.
| |
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Top
Abstract
Introduction
