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First published online March 20, 2009
doi: 10.1242/10.1242/dev.031377
1 National Centre for Biological Sciences, TIFR, GKVK Campus, Bangalore-65,
India.
2 Smurfit Institute of Genetics and TCIN, Lloyd Building, Trinity College
Dublin, Dublin-2, Ireland.
3 Department of Molecular and Cellular Biology, University of Arizona, Tucson,
AZ 85721, USA.
4 Department of Biological Sciences, Tata Institute of Fundamental Research,
Homi Bhabha Road, Mumbai-5, India.
* Authors for correspondence (e-mails: vijay{at}ncbs.res.in; veronica{at}ncbs.res.in)
Accepted 10 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Olfactory system, Neuronal activity, Gsk-3β (Shaggy), Wingless
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The mature brain also continues to change through turnover and reorganization
of neuronal branches and synapses. Here too, neuronal activity can effect
changes in circuit properties and connectivity, forming the basis for learning
and memory (Chklovskii et al.,
2004). Signals other than neuronal activity are also able to alter
mature neuronal circuits. For example, hormonal changes or stress can trigger
major changes in brain regions, including the death of neurons
(Truman and Riddiford, 2002)
or growth of neuronal branches (Lamprecht
and LeDoux, 2004). Thus, as in development, in the mature animal
neurotrophic factors and activity are both involved in effecting changes in
the brain. This plasticity of neural circuits in response to stimuli is
balanced by homeostatic mechanisms that allow circuits to yield the same
output in the face of random fluctuations in its activity or in environmental
factors (Marder and Goaillard,
2006). The adult brain therefore is sculpted by a range of
activity-dependent processes leading to synapse growth and elimination,
although the underlying mechanisms remain poorly studied, particularly in vivo
(Goda and Davis, 2003).
In this study, we ask whether the maintenance of neuron integrity is an
active process and, if so, what are the cell-autonomous and non-autonomous
mechanisms involved. In vertebrates, spontaneous activity functions to sculpt
the final aspects of circuit synaptogenesis and maturation at the glomerular
targets in the olfactory bulb (Yu et al.,
2004). In addition, activity in sensory neurons is required for
their maintenance, pointing to a relatively novel and unexplored
activity-dependent neuronal phenomenon (Yu
et al., 2004). We first established the Drosophila
olfactory system as a preparation in which mechanisms underlying neuronal
maintenance can be genetically analyzed in the context of its well-defined
network. The olfactory system in Drosophila is perhaps one of the
best-understood neural circuits in terms of its anatomy and function. There
are 
1200 olfactory sensory neurons (OSNs) that project from the sense
organs, which are located on the antennae and maxillary palps, to glomeruli
within the antennal lobe (see Fig.
1A) (Laissue and Vosshall,
2008). Within each glomerulus, OSNs that express a specific
odorant receptor (OR) gene synapse with excitatory and inhibitory local
interneurons and projection interneurons that wire to the mushroom bodies and
lateral protocerebrum. Such matching of OR expression in subsets of OSNs with
defined second-order neurons to create a functional glomerular specificity has
been observed in all insects and vertebrates examined
(Komiyama and Luo, 2006).
Here, we first confirm that, as in vertebrates
(Yu et al., 2004), neural
activity is necessary to maintain the integrity of adult Drosophila
OSNs and then extend this knowledge by detailed characterization of temporal,
cellular and molecular aspects of OSN maintenance. Cell-autonomous OSN
phenotypes resulting from a lack of activity can be mimicked by inhibiting
Wingless (Wg)/Wnt signaling and can be rescued by downregulating Gsk-3β,
overexpressing Dishevelled (Dsh) or Wg, or by increasing the endogenous
activity in OSNs. These and other observations indicate that neural activity
and the Wg/Wnt signaling pathway collaborate to regulate Gsk-3β activity
in the maintenance of OSN integrity. In mammals, the activation of Gsk-3β
has been demonstrated to lead to Alzheimer's disease-related synaptic
impairments (Liu et al., 2003;
Zhu et al., 2007). We discuss
the mechanisms and significance of this activity-dependent maintenance of the
olfactory peripheral sensory map that acts through Gsk-3β.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Larsson et al., 2004) were
provided by Leslie Vosshall (Rockefeller University, New York, USA),
UAS-IMPTNT-V, UAS-TNT-G by Sean Sweeney and Cahir O'Kane
(University of York, UK and University of Cambridge, UK),
UAS-Kir2.1-EGFP by Richard Baines (University of Manchester, UK),
UAS-2xEGFP by Haig Keshishian (Yale University, USA),
UAS-Wlds by Marc Freeman (University of Massachusetts,
USA), UAS-dsh DN.
DIX by Jeffrey Axelrod (Stanford University,
USA) and UAS-arrow C from Jean-Paul Vincent [National
Institute for Medical Research (MRC), UK]. UAS-eag-DN,
UAS-Sh-DN is a double recombinant of previously described strains
(Broughton et al., 2004;
Mosca et al., 2005) from
Subhabrata Sanyal. UAS-p35, UAS-sgg DN.A81T, UAS-sgg
CA.S9A, UAS-Axn-GFP, UAS-shibireK44A,
UAS-mCD8::GFP, UAS-pan.dTCFN, UAS-arm.S10,
FRT19A, FRT19A tub-Gal80 hsFlp, UAS-dsh-myc, UAS-wg-HA,
UAS-UBP2 and
tub-Gal80ts,tub-Gal80ts recombinant
stocks were obtained from the Drosophila Stock Center, Bloomington,
IN, USA.
Immunohistochemistry
Dissection and antibody staining of adult brain was carried out as
described previously (Jhaveri et al.,
2000). Primary antibodies used were 1:10 anti-Brp (DSHB), 1:50
mAb22C10 (DSHB), 1:10,000 rabbit anti-GFP (A6455, Molecular Probes), 1:1
anti-Wg (4D4, DSHB). Secondary antibodies used were Alexa 488 goat
anti-rabbit, Alexa 568 goat anti-mouse and Alexa 647 goat anti-mouse (1:200,
Molecular Probes). Labeled samples were mounted in 80% glycerol and imaged on
an Olympus Fluoview FV1000 confocal microscope at 1 µm intervals. Data were
processed using ImageJ and pseudo-coloring and enhancements were implemented
with Adobe Photoshop CS2. In all images, the right-hand antennal lobe is
shown, with dorsal up and lateral to the left.
In order to test for Wg release, dissected brains were subjected to spaced
5x K+ depolarization
(Ataman et al., 2008) by
dissecting in normal HL3-saline (Stewart
et al., 1994), then exposing to 90 mM KCl-HL3 solution for 5
minutes with gaps of 15 minutes in normal HL3-saline. Brains were fixed
immediately after treatment and stained with anti-Wg antibody as described
above. Pixel intensities were measured as described below and levels of
immunoreactivity represented as a `heat map'.
Generation of MARCM clones
Late second instar larvae of a cross between FRT19A, tub-Gal80,
hsFlp; UAS-TNT-G/CyO and FRT19A;
Or47b-Gal4, UAS-mCD8::GFP/CyO (60-72 hours AEL) were
heat shocked for 1 hour at 37°C to induce OSN clones that express
mCD8::GFP and TNT-G and were compared with controls that expressed only
mCD8::GFP.
Quantitative confocal imaging
Samples were imaged at 1 µm thickness at a frame size of 512x512
pixels under identical acquisition settings. Glomerular identification was
aided by staining with anti-Brp and compared with glomerular maps
(Couto et al., 2005;
Laissue et al., 1999). To
measure glomerular volume, contours of VA1v and VA6 were traced in individual
sections (see Fig. S4H in the supplementary material), and volumes calculated
by summation of individual areas in each section multiplied by the section
thickness. GFP intensity was estimated in regions of interests (ROIs) (see
green circles in Fig. S4F in the supplementary material) within the VA1v
glomerulus. To quantify the pixel footprint of OSN terminals, a modification
of a previously described method was used
(Brown et al., 2006). ROIs in
the GFP channel were traced to obtain the total number of pixels and the
intensity of each pixel. The background fluorescence intensity in the GFP
channel was used to set the threshold to determine the total number of pixels
showing GFP.
All measurements were manually traced with a virtual instrument written in LabVIEW 6.1 software (National Instruments). The values obtained were statistically analyzed, compared and plotted as histograms using the Origin 6.0 program (Microcal Software).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Neuhaus et al., 2005). These
heterodimers have recently been shown to form ligand-activated channels
(Sato et al., 2008;
Wicher et al., 2008). As
expected, neurons lacking Or83b show no odor-evoked activity and a strongly
diminished spontaneous activity (Larsson
et al., 2004). These mutants therefore provide a system for
investigating the role of neuronal activity during the development and
maintenance of OSNs.
We examined the morphology of OSNs in mutants null for Or83b by
driving green fluorescent protein (GFP) using Or83b-Gal4
(Or83b-Gal4, UAS-mCD8::GFP;
Or83b-/-). Or83b-Gal4 drives GFP in
80% of OSNs, allowing visualization of their sensory terminals in
antennal glomeruli (Fig. 1B)
and their axons in the outer nerve fiber layer
(Fig. 1B'). In
Or83b nulls, OSNs are indistinguishable from those of the wild type
at emergence (data not shown), supporting previous conclusions that OSN
targeting in Drosophila does not require neuronal activity
(Dobritsa et al., 2003). This
pattern remains unchanged up to 2 days post-eclosion (PE;
Fig. 1C), but older mutant
animals show defects in morphology of the OSNs, a phenotype clearly visible
within 6 days PE (Fig. 1D). The
sensory terminals (arrowheads in Fig.
1D) as well as axons (arrowheads in
Fig. 1D') appear
`beaded', or `blebbed', which are characteristics of axonal degeneration
(Saxena and Caroni, 2007). We
ruled out the possibility that the phenotypes are an artifact of a failure of
GFP transport into the axons (Larsson et
al., 2004) by staining brains for the neuron-specific
microtubule-associated protein Futsch, which is recognized by the monoclonal
antibody 22C10 (Fujita et al.,
1982; Hummel et al.,
2000). In these preparations, the axons in the outer nerve fiber
layer were apparent as broad tracts running from the antennal nerve entry
point to the antennal commissure in controls (see Fig. S1A in the
supplementary material), and these were severely disrupted in experimental
(Or83b-null) animals (see Fig. S1B in the supplementary
material).
We focused on a subset of OSNs (23-25) marked by Or22a>GFP
that converge onto the DM2 glomerulus (Fig.
2A,A'). As with Or83b>GFP discussed above, these
neurons were not detectably affected in 2-day-old mutants
(Fig. 2B), with signs of
degeneration becoming apparent by 4 days PE (arrow in
Fig. 2C), and progressing
further at 6 (Fig. 2D) and 8
(Fig. 2E) days PE, when the
number of axons innervating the glomerulus was reduced compared with 2 days
PE. This suggests that some axons are completely degenerated by 6 and 8 days
PE, which was confirmed by measuring the pixel intensity in regions over the
axon fascicles (see Fig. S2A-F and Table S6 in the supplementary material)
(P<0.005). By 6 days PE, the cell bodies were still unaffected
(see Fig. S2G-K in the supplementary material) (P>0.05),
indicating that degeneration initiates at the terminals and progresses in a
retrograde manner.
In order to test whether temporal expression of the Or83b protein could
rescue these defects, we exploited the Gal80ts TARGET system
(McGuire et al., 2003). Flies
of genotype Or22a-Gal4, UAS-mCD8::GFP/UAS-Or83b;
tub-Gal80ts,
Or83b-/tub-Gal80ts,
Or83b- were reared at 18°C (Or83b-OFF) and shifted to
29°C (Or83b-ON) at different times after eclosion. Phenotypes were
examined at 8 days PE (Fig. 2F)
and compared with age-matched mutants (Fig.
2E). Expression of the Or83b transgene beginning at
eclosion (not shown) or 2 days PE resulted in a significant rescue of the
defective phenotype when examined at 8 days
(Fig. 2F, compare with 2E). The
extent of rescue was quantified by estimating the pixel footprint of axonal
terminals within the DM2 glomerulus (Brown
et al., 2006). When targeted expression was initiated later (4
days PE), the rescue in glomerular arborization was also significant, although
some blebbing in the axons still occurred
(Fig. 2F',G; see Table S1
in the supplementary material) (P<0.005). Or83b expression
beginning 6 days PE could not rescue the phenotype
(Fig. 2F',G; see Table S1
in the supplementary material) (P>0.05) because significant
degeneration of neurons had already occurred. These results confirm a role for
Or83b function in stabilizing adult neurons; replacement of the wild-type
protein in mutants arrests further degeneration but cannot reverse effects
that have already occurred. The current reagents do not allow us to delineate
a critical period for Or83b requirement, if one exists; keeping Or83b on
during development, but not after eclosion, prevented the degeneration
normally observed at 6 days PE in mutants (see Fig. S3 and Table S7 in the
supplementary material). However, these results are difficult to interpret
given the presumed slow kinetics of the Gal4/Gal80ts system coupled
with the undetermined stability of the Or83b protein.
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), we examined whether the Or83b-null mutant
phenotype could be rescued by induction of activity. Transgenic expression of
dominant-negative (DN) ether a go-go (eag) or
Shaker (Sh) K+ channels have been shown to
greatly enhance membrane excitability in neurons
(Broughton et al., 2004;
Mosca et al., 2005). We
targeted expression of Eag-DN and Sh-DN channels into the
Or22a-Gal4-expressing OSNs in Or83b-null mutant animals. Animals of
the genotype Or22a-Gal4, UAS-mCD8::GFP/UAS-eag-DN,
UAS-Sh-DN; Or83b-/-, were generated and the
status of OSNs examined 6 days PE (Fig.
2H). A comparison of these neurons with those of mutants at the
same age (Fig. 2D) showed a
significant rescue of the defective phenotype, although some beading was still
apparent in the axons; the extent of the terminal pixel footprint was
comparable to that of normal animals (Fig.
2A',G; see Table S2 in the supplementary material)
(P>0.05). We independently confirmed that ectopic expression of
mutant K+ channels did not result in altered terminal arborizations
in wild-type neurons due to activity-dependent synaptic changes
(Fig. 2G; see Table S2 in the
supplementary material) (P>0.05). These observations support the idea that neuronal activity, probably triggered by Or83b, is necessary for OSN survival in the adult. This suggests that blocking neuronal activity by other means might have effects similar to that seen in Or83b mutants.
Neuronal activity and synaptic function are necessary for OSN stability in the adult olfactory system
The human inward rectifier K+ channel (Kir2.1; KCNJ2
- HUGO) has been shown to hyperpolarize neuronal membranes, thereby inhibiting
the generation of an action potential
(Baines et al., 2001). Flies
carrying a UAS-Kir2.1 transgene were crossed with
Or83b-Gal4, UAS-2xEGFP,
2xtub-Gal80ts or Or22a-Gal4,
UAS-mCD8::GFP, 2xtub-Gal80ts. Progeny were
reared at 18°C until adulthood (Kir2.1-OFF). Newly eclosed
flies were transferred to 29°C for 6 days (Kir2.1-ON), brains
dissected and stained with antibodies to GFP and with the monoclonal antibody
nc82, which recognizes the presynaptic protein Bruchpilot (Brp)
(Wagh et al., 2006). OSNs
`silenced' by Kir2.1 for 6 days PE showed a disruption of the
glomerular pattern (Fig. 3B
compared with 3A). We confirmed
that the pattern was normal upon eclosion and until 3 days PE (data not
shown). This phenotype could also be generated in a smaller subset of
Or22a-Gal4-expressing neurons (Fig.
3G-G''' compared with
3F). The `beaded' axon
phenotype was apparent at 3 days PE (Fig.
3G) and became progressively more severe by 5 days PE
(Fig. 3G''').
Silencing neurons by expression of Kir2.1 inhibits the
generation of action potentials, thus compromising both axonal conduction and
synaptic release. We directly perturbed synaptic function by expression of a
mutant Shibire protein (ShiK44A) that blocks synaptic vesicle
recycling (Moline et al.,
1999), or of the light chain of tetanus toxin (TeTxLC), which acts
to enzymatically cleave neuronal synaptobrevin (n-Syb), thus abolishing evoked
neurotransmitter release and causing a significant reduction in spontaneous
release (Baines et al., 1999;
Sweeney et al., 1995).
Adult-specific expression of either ShiK44A (Fig. 3C) or TeTxLC (TNT-G) (Fig. 3E,E') using the Or83b-Gal4 driver disrupted the integrity of OSNs, phenocopying the Or83b-null mutant defect. Degeneration was detected after 3.5 days PE (Fig. 3E) and became progressively more severe as the animal aged (Fig. 3E'). The cell bodies of the OSNs, however, were unaffected even after 6 days of treatment (see Fig. S2I-K in the supplementary material; P>0.05).
In these experiments, because we co-expressed GFP to monitor the status of the OSNs this lead to a possible concern that the phenotypes seen were artifacts of potentially reduced GFP levels when another transgene is introduced. The following observations argue against this possibility. First, there was no significant difference (see Fig. S4E and Table S8 in the supplementary material) (P>0.05) in the average fluorescence intensity of pixels highlighted by the GFP reporter in preparations in which a mutated and inactivated tetanus toxin (IMPTNT-V) was expressed (see Fig. S4A in the supplementary material) as compared with TNT-G (see Fig. S4C in the supplementary material). Second, the volume of VA1v, the target of Or47b-Gal4-expressing neurons, when expressed relative to that of the unaffected neighboring glomerulus VA6, was significantly reduced (see Fig. S4G and Table S8 in the supplementary material) (P<0.005) when TNT-G (see Fig. S4D in the supplementary material) but not IMPTNT-V (see Fig. S4B in the supplementary material) was expressed. Lastly, we confirmed that expression of TNT-G by Or83b-Gal4 disrupted the architecture of the nerve fiber layer by staining brains with mAb22C10 (see Fig. S1D compared with controls in S1C in the supplementary material).
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Activity is autonomously required in OSNs for their maintenance and glial cells are able to respond to degenerating neurons
The requirement for normal synaptic release for maintenance of neurons
raises the possibility that active neurons release neurotrophic factors
required for survival. In such a scenario, we would expect these signals to be
non-autonomous when the blocking of activity in a subset of neurons could be
compensated by active neighbors. Above, we demonstrated that silencing OSNs
that innervate a single glomerulus in an otherwise normal antennal lobe led to
degeneration of these neurons (Fig.
3G-G'''). Hence, non-autonomous cues, if any, cannot be
transferred across glomeruli. We used the MARCM
(Lee and Luo, 2001) technique
to generate clones of subsets of silenced OSNs within a population of cells
innervating a single glomerular target. Recombination induced by a heat
shock-regulated Flip-recombinase (hsFlp) results in expression of
TNT-G in a subset of neurons within the domain of expression of
Or47b-Gal4 that targets the VA1v glomerulus. Control
(FRT19A/FRT19A, tub-Gal80, hsFlp; Or47b-Gal4,
UAS-mCD8::GFP/+) and experimental (FRT19A/FRT19A tub-Gal80
hsFlp; Or47b-Gal4 UAS-mCD8::GFP/UAS-TNT-G) siblings
with 10-12 out of 45 neurons `silenced' are shown in
Fig. 4. OSNs expressing TNT-G
showed severe degeneration with age, and by 7 days PE only a few terminals
were visible (Fig. 4B,D,F). We
obtained a few clones in which an even smaller number of OSNs were silenced
(see Fig. S5 in the supplementary material). In all these preparations,
including clones in which only two cells were silenced (see Fig. S5C in the
supplementary material), neurons degenerated despite the fact that the
neighboring cells were normal. Together, these data strongly argue that OSNs
have an autonomous requirement for activity to ensure their survival.
Caspase-dependent pathways of programmed cell death do not mediate the
neurodegeneration we observe because co-expression of the pan-caspase
inhibitor, baculovirus p35, did not alter the lesions induced by TNT-G or
Kir2.1 (see Fig. S6B,E compared with S6A,D in the supplementary
material). The suggestion of a mechanism of axonal degeneration that is
independent of cell death (Saxena and
Caroni, 2007) is supported by the observation that axonal
degeneration in Or83b-null animals in which activity was silenced did
not involve the OSN soma, even 6 days PE (see Fig. S2G,H in the supplementary
material) (P>0.05).
Studies by Freeman and colleagues
(MacDonald et al., 2006)
demonstrated that transection of the antennal nerve results in a process
resembling the Wallerian degeneration elucidated in vertebrates. The
Wlds transgene, which protects neurons against Wallerian
degeneration, could not, however, rescue the phenotypes obtained upon TNT-G or
Kir2.1 expression (see Fig. S6C,F compared with S6A,D in the
supplementary material). The degeneration does, however, require the
proteasome degradative machinery because expression of the yeast
ubiquitin-specific protease UBP2
(DiAntonio et al., 2001;
Watts et al., 2003) protected
Or22a neurons from degeneration in the Or83b-null background (see
Fig. S6G-I and Table S4 in the supplementary material)
(P<0.005).
Subsequent to degeneration, axon terminals were `cleared' such that only
GFP-marked remnants remained by 7 days PE
(Fig. 4F). The levels of the
glial phagocytic receptor Draper
(MacDonald et al., 2006) were
elevated in these regions (see Fig. S6N in the supplementary material).
Animals in which Or47b neurons were silenced (Or47b-Gal4,
UAS-mCD8::GFP/UAS-TNT-G;
2xtub-Gal80ts/+ shifted to 29°C) exhibited
higher levels of Draper in the target glomerulus, VA1v, than controls
(Or47b-Gal4, UAS-mCD::GFP/UAS-IMPTNT-V;
2xtub-Gal80ts/+) (see Fig. S6J-N and Table S8
in the supplementary material) (P<0.05). Staining with the
glial-specific marker Repo did not reveal an increase in cell number (data not
shown). This means that extant glial cells upregulate Draper expression in
response to degenerating OSNs.
|
).
Further, altered Gsk-3β activity has been implicated in disease models of
neural degeneration (Liu et al.,
2003; Zhu et al.,
2007). We reasoned that if neuronal activity impinged upon
Gsk-3β, then inhibiting its activity could bypass the requirement for
neuronal activity. A dominant-negative Gsk-3β construct (UAS-sgg
DN.A81T) (Ataman et al.,
2008; Bourouis,
2002) was targeted to the Or22a OSNs in Or83b homozygous
null animals. When examined at 6 days PE, axons as well as presynaptic
terminals showed significant rescue as compared with age-matched controls
lacking the transgene (Fig. 5B
compared with 5A,D; see Table
S3 in the supplementary material) (P<0.005). Hence, inhibition of
Gsk-3β can compensate for neural activity and preserve neuronal
integrity. Lithium chloride (LiCl) is a known pharmacological blocker of
Gsk-3β activity (Kirshenboim et al.,
2004). Feeding Or83b-null mutants with a 10 mM LiCl diet
from immediately after eclosion resulted in a considerable rescue (Fig.
5C compared with
5A,D (see Table S3 in the
supplementary material) (P<0.005).
These observations indicate that neuronal activity mediates its effect by
inhibiting the kinase activity of Gsk-3β. Consistent with such a model,
constitutive activation of Gsk-3β resulted in phenotypes resembling that
resulting from a lack of neuronal activity. Driving constitutively activated
Gsk-3β (UAS-sgg CA.S9A)
(Bourouis, 2002) for 6 days PE
in Or22a, Or47b and Or83b adult OSNs using the inducible Gal80ts
system (Fig. 5E-G) resulted in
significant degeneration of presynaptic terminals (dotted lines in
Fig. 5
E',F',G'compared with
5E,F,G) and axons. Gsk-3β
is a known sensor for several different signaling pathways; regulation via the
Wnt pathway is mediated by the cytoplasmic phosphoprotein Dsh, which signals
to the Axin-Apc-Gsk-3β complex (Logan
and Nusse, 2004). Ectopic expression of Dsh
(Penton et al., 2002)
protected the Or22a OSNs of Or83b-null animals from degeneration,
even in 6-day-old animals (Fig.
5H,H'; see Table S4 in the supplementary material)
(P<0.005). It is known that increased levels of Dsh can trigger
Wnt signaling and lead to an inhibition of Gsk-3β activity
(Ciani et al., 2004). This
provides further evidence for the role of Wnt signaling in conferring neuronal
stability.
|
). When expressed in the Or22a OSNs (w; Or22a-Gal4,
UAS-mCD8::GFP/UAS-GPI-fz+;
tub-Gal80ts, tub-Gal80ts/+), or in Or83b
OSNs during adulthood, axons and their terminals showed characteristic signs
of degeneration (Fig. 6Ab,Bb
compared with 6Aa,Ba). Further,
perturbation of Arrow co-receptor function by expression of a truncated
protein (Arrow C) (Fig.
6Ac,Bc) (Piddini et al.,
2005), or a dominant-negative (DN) form of dsh (dsh
DN.DIX) (Fig.
6Ad,Bd) (Axelrod et al.,
1998), or by overexpression of Axin
(Cliffe et al., 2003)
(Fig. 6Ae,Be) also caused
neurodegeneration.
In the canonical Wg/Wnt pathway, the Axin-Apc-Gsk-3β complex acts to
stabilize β-catenin, leading to transcriptional effects
(Logan and Nusse, 2004). In
our studies, expression of an activated form of β-catenin [activated
Armadillo (Arm)] (Pai et al.,
1997) (Fig. 6Af,Bf)
or of a dominant-negative form of Drosophila TCF (dTCF; Pangolin)
(DN-dTCF) (Fig. 6Ag,Bg)
(van de Wetering et al., 1997)
for 6 days did not compromise neuronal stability. These experiments together
indicate that signaling downstream of Gsk-3β/Axin does not occur through
transcriptional events mediated by dTCF. The end-point of Wg/Wnt signaling is
therefore likely to be non-transcriptional and to involve a cytoplasmic role
of Gsk-3β in regulating axon stability. This mechanism requires detailed
investigation.
These results together lead to the hypothesis that Wnt signaling through
Gsk-3β confers neuronal stability, which could have effects at synapses
through the divergent canonical pathway
(Ataman et al., 2008;
Miech et al., 2008;
Salinas, 2007;
Speese and Budnik, 2007). Is
there a link between neuronal activity and Wg signaling? Immunostaining of
adult antennal lobes showed low Wg levels in glomeruli
(Fig. 7A; see Fig. S7 in the
supplementary material). Global activation of neurons by KCl-induced spaced
depolarization (Ataman et al.,
2008) led to a significant increase in Wg levels within the
antennal glomeruli (Fig. 7B
compared with 7A; see Table S5
in the supplementary material) (P<0.005). These data do not
provide sufficient resolution to decipher whether Wg is released from the OSNs
themselves or non-autonomously from the antennal lobe interneurons as a
consequence of their activation.
|
) in the Or22a OSNs in Or83b-null animals during
adulthood and examined animals 6 days PE
(Fig. 7E). Careful examination
of the footprint of the Or22a terminals in the DM2 glomerulus indicated
significant rescue of the degeneration as compared with control animals
(Fig. 7D; see Table S4 in the
supplementary material, plotted in Fig.
7F) (P<0.005). This suggests that autocrine signaling
by Wg does, at least in part, confer neuroprotection to the OSNs. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Spitzer, 2006). In the mature
vertebrate nervous system, many neurons are eliminated, although the majority
survive through the life of the animal. Little is known about the mechanisms
underlying the maintenance of mature neurons in a circuit, or, indeed, whether
there is any active requirement at all for maintenance. Given the fundamental
role of electrical activity in neuronal function, we asked whether it is the
sensor for the integrity of the nerve cell. In order to test this rigorously,
we needed to have a preparation from which both spontaneous and evoked
activity could be removed and the consequences on neuronal maintenance
assessed. The OSNs are unique in that they allow the manipulation of
spontaneous and odor-induced activity in the mature animal. Our experiments in
this system implicate a role for activity in neuronal maintenance. Further, we
show that Gsk-3β, a molecule that is a sensor for nutrients and Wnt
signals (Grimes and Jope,
2001), acts `downstream' of neuronal activity in this process. Our
results thus integrate a key property of neurons, electrical activity, with
Gsk-3β signaling (summarized in Fig.
7G). Although Gsk-3β can receive inputs from a variety of
pathways, the expression of Wg in the adult antennal lobe and the observation
that degeneration could be rescued by expression of Dsh and Wg, suggested that
this is a major signaling pathway acting on neuronal survival in the mature
animal. We also established that Wnt signaling acts through a
non-transcriptional pathway involving Gsk-3β. The action of Wnt signaling
during the formation and plasticity of neuronal circuits is mediated through
the `divergent canonical pathway', as demonstrated by several studies in both
vertebrates and Drosophila (Ataman
et al., 2008; Hall et al.,
2000; Miech et al.,
2008; Packard et al.,
2002).
Neuronal activity in the maintenance of integrity: autonomy at the sub-glomerular level
Gogos and colleagues have demonstrated that spontaneous activity is
essential for both the development and maintenance of OSN projections in the
mouse olfactory bulb (Cao et al.,
2007; Yu et al.,
2004). In Drosophila, the formation of the peripheral
olfactory map is independent of ORs or activity, and is possibly hardwired
(Dobritsa et al., 2003).
However, as in mammals, OSN maintenance requires neural activity. We have
exploited the availability of Or83b-null mutants and the conditional
TARGET system (McGuire et al.,
2003) to demonstrate that OSN terminals within the antennal lobe
glomeruli develop normally in the absence of activity, but show local
degeneration in older animals in which the most distal ends exhibit signs of
beading, blebbing and, eventually, fragmentation, which are hallmarks of axon
degeneration (Saxena and Caroni,
2007).
In vertebrates, electrically silent visual system neurons
(Hua et al., 2005) and OSNs
(Zhao and Reed, 2001) retract
when placed in an environment of active neurons because of synaptic
competition for target sites. When all neurons in a given field are silenced
to the same extent, elimination does not occur, suggesting that differences in
activity, rather than the absolute activity state, determine the stability of
connections. Contrary to expectation from these findings, neurons innervating
a single glomerulus retracted their contacts and showed degeneration when
silenced. Further, clones that drive TNT-G in small subsets of OSNs also
degenerate, indicating that activity influences neuron survival by exerting
autonomous effects at the level of individual cells.
Gsk-3β as a key sensor of inputs for neural activity and Wnt signaling in maintaining circuit stability
Enhanced neuronal activity has been shown to trigger the activation of a
wide range of genes including transcription factors, cell adhesion molecules,
membrane excitability proteins (Guan et
al., 2005), translational regulators such as dFmr (Fmr1)
(Tessier and Broadie, 2008)
and signaling molecules such as Wnt (Ataman
et al., 2008). Our observation that Wg is expressed in the adult
brain led us to test the role of Wg/Wnt signaling in stability. We found that
downregulation of Wg pathway members compromises OSN stability, resulting in
phenotypes similar that resulting from a lack of neuronal activity. Several
studies argue for a link between neuronal activity and Wnt/Wg signaling during
formation of LTP (Ahmad-Annuar et al.,
2006; Chen et al.,
2006) and in activity-dependent dendritic morphogenesis
(Wayman et al., 2006;
Yu and Malenka, 2003). At the
Drosophila neuromuscular junction, activity-dependent Wg secretion
results in structural outgrowth mediated by Gsk-3β in the motoneurons and
nuclear localization of the cleaved C terminus of DFz2 in the postsynaptic
muscle cells (Ataman et al.,
2008).
We have provided evidence for a requirement of Wg/Wnt signaling for
stabilization of adult OSNs, although a transcriptional output of the pathway
is not required. Non-transcriptional roles for Wnt signaling have been
demonstrated previously, well-studied examples being in Wnt7a-induced growth
cone and axon remodeling of the mossy fibers
(Hall et al., 2000) and in
Drosophila neuromuscular junction synaptogenesis and plasticity
(Ataman et al., 2008;
Packard et al., 2002). The
output of signaling in these systems is mediated by Gsk-3β, which
regulates microtubule cytoskeletons. Gsk-3β phosphorylates the
Microtubule-associated protein 1B (mammalian homolog of Futsch) and Tau,
thereby influencing microtubule stability
(Goold and Gordon-Weeks,
2004). Neuronal activity regulates Gsk-3β enzymatic activity
through a series of phosphorylation and dephosphorylation events
(Hooper et al., 2007;
Peineau et al., 2007), whereby
activity-regulated PP1 phosphatase and PI3K-Akt kinase regulate
phosphorylation of Gsk-3β serine 9.
Activation of Gsk-3β in rat hippocampus inhibits LTP with associated
synaptic impairments reminiscent of Alzheimer's disease
(Liu et al., 2003;
Zhu et al., 2007). A genetic
link between late-onset Alzheimer's disease and the Wnt pathway co-receptor
LRP6 has been demonstrated in human subjects
(De Ferrari et al., 2007) and
there is a possibility that Alzheimer's disease-associated synaptic
impairments are due to aberrant GSK-3β kinase activity
(Liu et al., 2003;
Zhu et al., 2007). The
phenotypes we observed in OSNs with activated Gsk-3β or a chronic
blockage of activity are tantalizingly similar to those described during
neurodegeneration in Alzheimer's disease models
(Inestrosa and Toledo, 2008;
Toledo et al., 2008).
We propose a model whereby neuronal activity acts together with Gsk-3β
to maintain circuit stability in the adult olfactory system
(Fig. 7G). Activity appears to
lead to Wg release, which acts in an autocrine manner to impinge upon
Gsk-3β activity. It is also possible that autonomous activity could
signal to Gsk-3β through other pathways, one of them being the energy
status of the neuron (Grimes and Jope,
2001). In adult OSNs, Wnt signaling appears to be
non-transcriptional and Gsk-3β possibly acts cell-autonomously to
regulate the dynamics of the microtubule cytoskeleton
(Salinas, 2007). Our results
support the emerging view of a role for the Wnt pathway in neuroprotection,
and our approach provides a system in which to examine the structural and
molecular mechanisms that operate during altered physiological states in a
genetically tractable organism.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1273/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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