Transcript Document

OCTOPAMINE-DEFICIENT MUTATION SUPPRESSES SHAKER PHENOTYPES IN DROSOPHILA
P.K.Rivlin, J.L. Krans, K.D. Gawera, S.W. Wong and R.R.Hoy. Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853
OVERVIEW
• We are using Drosophila mutants with altered levels of bioamines (in
particular, octopamine, dopamine, serotonin) to investigate the role that
bioamines play in controlling behavior, synaptic development/function
and lifespan.
• It is well established that ion channel mutants alter neural activity and
display a number of behavioral, developmental and physiological
phenotypes.
• Using double mutant combinations, we are investigating how bioamines
contribute to the phenotypes observed in ion channel mutants, and whether
bioamines play a role in the homeostatic regulation of activity at the nerve
terminal.
2. Oxidative stress resistance is increased in
M18
5
Tßh
Sh double mutant.
Genotype
% Survival
n
CS
TßhM18
TßhM18 Sh5
Sh5
eag Sh133
90.0
95.8
87.5
51.3
30.0
70
230
120
230
187
5. Synaptic physiology of wildtype and Sh5
Figure 2. Suspectibility to 10 mM paraquat, a compound that generates free-radicals. Order of severity
correlates with lifespan: eag Sh133 < Sh5 < TßhM18 Sh5 < TßhM18 ≈ CS. The hyperexcitable mutant, eag
Sh133 serves as a control with severely reduced lifespan.
3. Genetic screen for suppressors of Shaker
Shaker (Sh) potassium channel mutants display a number of behavioral
and physiological phenotypes that include leg-shaking under ether anesthesia
(Kaplan and Trout 1969), altered larval locomotion (Wang et al. 2002),
repetitive firing of action potentials in larval nerves, prolonged
neurotransmitter release at the larval neuromuscular junction (Jan et al. 1977;
Ganetzky and Wu 1982), and reduced lifespan (Trout and Kaplan 1970).
These phenotypes are enhanced when Sh is combined with another
potassium channel mutant, ether-a-go go (eag). In addition, expansion of
octopamine-containing nerve terminals (type II) is observed at the
neuromuscular junction in eag Sh mutants (Budnik et al. 1990). This raises
the possibility that octopamine may contribute, in part, to the enhanced
phenotypes observed in eag Sh. To address this question, we have begun an
analysis of Sh mutants with genetically altered octopamine levels. Here we
present an analysis of a TßhM18 Sh5 double mutant. TßhM18 is a null mutation
in the tyramine beta hydroxylase gene and is unable to synthesize
octopamine (Monostirioti et al. 1990).
1.
M18
Tßh
partially rescues lifespan in
100
5
Sh
mutant.
Tßh[M18]
Tßh[M18]Sh[5]
Sh[5]
90
[C155-GAL4] eag Sh133
EP Line
(UAS-gene X)
Figure 5. Evoked potentials (ejps) from wildtype and Shaker genotypes. Right: Sh5 mutant Left:
CantonS strain. Each trace is the average of 20 repetitions of electrical stimulation to the segmental nerve
in third instar larval preparations. Voltage recordings are acquired in modified Standard Saline (Jan and
Jan 1976) using a single sharp electrode . Mean amplitude of Sh5 ejps was always greater than that of CS,
and this relationship was heavily [Ca++] dependent.
Note: Since Jan, Jan and Dennis (1977) reported these differences at the NMJ, several new salines have
been developed for the larval preparation. It is our experience that these at least partially mask the Sh5
NMJ phenotype, even at lowered [Ca++], where the magnitude difference is typically greater than 10-fold.
suppressor
no effect
enhancer
6. TßhM18 suppresses the Sh5 NMJ phenotype.
40
“increased stress resistance”
Figure 3. EP misexpression screen to identify genes that rescue the shortened lifespan of Shaker.
Briefly, an EP element is a transposable P element containing a basal promoter and 14 copies of the yeast
UAS sequence, which responds to the transcription factor GAL4. Each EP line is crossed to C155, a
transgenic fly that expresses GAL4 in all post-mitotic neurons. F1 flies are generated in which GAL4
drives high expression of the genes adjacent to the EP element. Each EP line has been sequenced and the
associated gene identified. F1 males are tested for resistance to paraquat. Thus far, we have identified one
putative suppressor, glutathione S1 transferase (GSTS1). GSTS1 is an enzyme involved in free-radical
homeostasis.
muscle 6
muscle 12
35
Mean EJP Amplitude (mV) _
Does octopamine contribute to Shaker phenotypes?
*
30
*
25
20
31.92
15
20.69
10
80
5
% survival
70
4. Larval Neuromuscular Junction (NMJ)
60
50
2.55
4.03
1.06
2.53
4.84
3.39
0
CS
Sh5
M18
M18Sh5
40
Figure 6. TßhM18 mutation suppresses Shaker phenotype. Recordings were made from both muscle 6 and
muscle 12 in larval preparations (number of muscle recordings per strain, left to right: 12, 7, 11, 6). In all
cases, muscle 12 showed smaller mean ejps than muscle 6 (not significant; p>0.05, all). TßhM18 (M18)
showed a non-significant increase in ejp amplitude over CS (m6: p>0.3, m12: p>0.05), but when combined
with the Sh5 mutation (M18Sh5), the double mutant abolished the increased ejp size that is characteristic
of Shaker mutants (Sh5) (*: p<<0.01). Insets: examples of voltage recordings from mutants not shown
above, TßhM18 and TßhM18Sh5, and CS. Scalebar: 25ms, 5mV.
30
20
10
0
1
4
7
10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76
days
Figure 1. Survival curves at 25o C. Sh5 (red, n = 571); TßhM18 Sh5 (green, n = 322); TßhM18 (blue, n = 376).
Sh5 displays a 43% reduction in mean lifespan as compared to TßhM18 (32.2 vs. 56.8 days). TßhM18 Sh5
displays a 26% extension in lifespan as compared to Sh5 (40.5 vs. 32.2 days). TßhM18 lives a normal lifespan
(unpublished observations).
CONCLUSIONS & FUTURE DIRECTIONS
Figure 4. Schematic of larval neuromuscular preparation and recording paradigm. Larval muscles are
shown in gray. The segmental nerve was stimulated to evoke synaptic potentials. Minimum intensity
stimuli to evoke full response were determined experimentally. Excitatory junctional potentials (EJPs)
were collected at muscles 6 and 12. Note that octopamine is present at the NMJ of muscle 12, but not at
muscle 6.
(1) Our analysis of TßhM18Sh5 suggests a link between synaptic transmission, oxidative stress and aging. In
addition, our finding that overexpression of GSTS1 in the CNS rescues stress resistance in Shaker
suggests that nervous system function is a critical determinant of aging.
(2) It is well established that cAMP plays a role in regulating synaptic function and development. The
absence of OA and/or the elevated levels of its precursor, tyramine may result in altered cAMP levels
which act to suppress neurotransmitter release as well as increase stress resistance in Sh5 mutants.
(2) To address the role of cAMP in aging, we are examining whether the cAMP mutants dunce (dnc) and
rutabaga (rut) affect lifespan and stress resistance.
(3) To address the role of synaptic transmission in aging, we are extending our studies to synaptic machinery
mutants.
(4) Lastly, we are investigating whether there is a critical period for lifespan rescue in TßhM18Sh5.
ACKNOWLEDGEMENTS
We thank Drs. M. Monostirioti and C-F Wu, and the Bloomington Stock Center for providing fly stocks.
This work was supported by a HHMI Professor Award to RRH.