Group II metabotropic glutamate receptors as potential pharmaceutical targets
for neurofibroma formation
Michael Stern, Ph.D.
William Marsh Rice University
Houston, TX 77005
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Group II metabotropic glutamate receptors as potential pharmaceutical targets for
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Michael Stern, Ph.D.
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William Marsh Rice University
Houston, TX 77005
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Several lines of evidence suggest that neurofibroma formation involves hyperactivation of PI3K within the glial layer that surrounds the motor neurons, and yet
the signals within gliby which PI3K activity is regulated remain incompletely understood. Following our recent discovery that the Drosophila group II
metabotropic glutamate receptor (DmGluRA) activates PI3K in motor neuron, we hypothesized that activation of DmGluRA might similarly activate PI3K in glia.
In task #1, we proposed to test if inhibition of DmGluRA-PI3K activity in motor neurons is sufficient to activate PI3K in the analogue of the Schwann cell called
the peripheral glia (as monitored by perineurial glial growth). We found that that inhibiting PI3K activity by introducing the DmGluRAI 12b null mutation or by
expressing the PI3KDN transgenin motor neurons did significantly increase perineurial glial growth. In task #2, we proposed to determine if DmGluRA activity
in peripheral glia is required for PI3K activation The stock construction required to address this task are almost complete and experiments aranticipated to
begin within the next few weeks.
15. SUBJECT TERMS
molecular genetics; neuroscience; cell biology; cell signaling; model systems model
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Table of Contents
Key Research Accomplishments.6
Several lines of evidence have suggested that neurofibroma formation occurs at least in
part via hyperactivation of PI3K, which is a direct target of Ras activity (Dasgupta et al., 2005;
Johanessen et al., 2005; Lavery et al., 2007). The involvement of PI3K in neurofibroma
formation suggests that molecules that participate in the regulation of PI3K in peripheral nerves
might be promising targets for therapeutic intervention. However, our understanding of the
mechanisms by which PI3K activity in the nervous system is regulated is incomplete. Recently,
my lab showed that in Drosophila larval motor neurons, PI3K is activated by the group II
metabotropic glutamate receptor DmGluRA (Howlett et al., 2008), raising the possibility that
antagonists of these group II receptors might prevent PI3K activation and thus act
therapeutically in neurofibroma formation. In this exploratory-hypothesis development award, I
proposed two tasks to extend these observations. First (task one), I proposed to determine if
inhibition of the DmGluRA-PI3K pathway in motor neurons would be sufficient to increase
growth of the outer, perineurial glial layer (analogous to the mammalian perineurium).
Preliminary results suggest that this inhibition does indeed increase perineurial glial size. Second
(task two), I proposed to determine if DmGluRA activity in peripheral glia (analogue of the
Schwann cell) is required for increased perineurial glia. The fly stocks required to perform this
task are almost finished, and the experiments to answer this question are expected to begin in the
next few weeks.
Task one: Does the increased motor neuron excitability conferred by inhibiting the
mGluRA-PI3K pathway promote perineurial glial growth?
In this aim, I proposed to inhibit DmGluRA-PI3K activity in motor neurons and, using
electron microscopy, monitor the resulting effects on perineurial glial thickness. I proposed to
perform these analyses both in a wildtype background, as well as in a background in which the
inebriated-e ncoded neurotransmitter transporter was eliminated by chromosomal mutation ( ine ').
The use of the ine mutation was previously shown to sensitize the peripheral nerve to trophic
effects of other mutations and to reveal effects on perineurial glial growth that were otherwise
difficult to demonstrate.
We found that combining the ine' mutation with genotypes that disrupt DmGluRA-PI3K
activity in motor neurons does indeed significantly increase perineurial glial thickness. In
particular, in ine mutants carrying the DmGluRA null mutation DmGluRA 112h , perineurial glial
thickness was increased from 1.27 +/- 0.1 to 2.05 +/- 0.2 pm (p=0.004, see Figure 1 below). In
addition, in ine mutants in which PI3K activity in motor neurons was blocked by D42-Gal4-
induced motor neuron expression of PI3K DN , perineurial glial thickness was increased to 1.68 +/-
0.15 pm. Although this effect is significant (p=0.048, see Figure 1 below), it is barely so and the
increase in glial thickness is less than what we observe in ine; DmGluRA 1121 '. Why? We have
recently obtained evidence from other experiments that the PI3K DN transgene is a considerably
weaker blocker of PI3K activity than the related transgene in which PTEN (phosphatase that
antagonizes PI3K) is overexpressed. Therefore I hypothesize that the weak phenotype of
Perineuria! glial thickness (|tm)
PI3K dn results from a weak transgene. This possibility will be tested by constructing and
analyzing larvae in which PTEN is overexpressed in an ine mutant background.
Figure 1: Perineurial glial thickness is increased in ine; DmGluRA inb double mutants. A)
Mean perineurial glial thickness (Y axis) +/- SEMs for the genotypes indicated along the X axis. The
following genotypes showed significant differences in glial thickness (Student’s t test): ine (lane #1,
n=18) versus ine; DmGIuRA 112h (lane 3, n=14), p=0.004. ine; D42>+ (lane #2, n=31 ) versus ine;
D42>PI3 K dn (lane #4, n=8), p=0.048. B) Representative transmission electron micrographs from
larval peripheral nerves of the indicated genotypes. Scale bars as indicated.
In the next funding year, we will analyze the same larvae as above, except in an ine +
background. I predict that this wildtype background will restore perineurial glial growth to
normal levels. We will also finish up measuring glial thickness in ine mutants overexpressing
Foxo + . This experiment has been delayed because the we picked up lethal mutations on the ine;
UAS-Foxo + chromosome but that problem has been solved and we are now ready to collect data.
Task two: Is mGluRA activity in peripheral glia required for the ability of motor neuron
activity to promote perineurial glial growth? This question is based on previous observations
from my lab that larvae doubly mutant for ine and second gene called push , which encodes an E3
ubiquitin ligase, also exhibit greatly increased perineurial glial thickness (Yager et al., 2001). I
hypothesize that the increased perineurial glial thickness in ine push double mutants occurs
because the ine push genotype increases glutamate release from motor nerve terminals, thus
hyperactivating PI3K in the peripheral glia via DmGIuRA. If so, then blocking DmGIuRA
specifically in peripheral glia is predicted to block this PI3K hyperactivation and thus decrease
perineurial glial thickness in ine push.
To address this hypothesis, I proposed to construct ine push larvae in which peripheral
glial DmGIuRA activity was knocked down via RNAi. To accomplish this task, we constructed
two stocks: in the first, we recombined the peripheral glial Gal4 driver gli-Gal4 onto the ine
push second chromosome, whereas in the second, flies carried ine push on the second
chromosome, and UAS-DmGluRA-RNAi on the third chromosome. Because push mutations
confer sterility, the second chromosomes of both stocks are balanced with the CyO balancer
marked with GFP (to enable us to distinguish homozygous from balanced larvae). We suffered
from the usual problems of chromosomes picking lethal mutations, which prevents acquisition of
homozygous larvae. This problem delayed construction of the necessary stocks but now we
have the two stocks we need that will produce the desired larvae. We are now poised to cross
together flies from the two stocks, and perform electron microscopy on the non-GFP labeled
third instar larval progeny. In the next funding year, these larvae will be analyzed, and we will
determine if DmGluRA expression specifically in peripheral glia is sufficient to rescue the glial
growth phenotype of ine push ; DmGluRA 1121 '.
KEY RESEARCH ACCOMPLISHMENTS
In preliminary results, we have found that blocking DmGluRA-PI3K activity in motor
neurons in an ine mutant background increases perineurial glial thickness. However, this
increased thickness is less extreme than the increased thickness observed in the ine push double
mutant. These observations support the hypothesis originally put forth, but raise the possibility
that regulators in addition to the neuronal DmGluRA-PI3K pathway play roles in the control of
perineurial glial thickness.
The observations that blocking DmGluRA-PI3K activity in motor neurons in an ine mutant
background increases perineurial glial thickness support the hypothesis originally put forth.
However, the increase that we observe is much less than what we observed in the ine push double
mutant, which was the phenotypic basis for the hypothesis. Therefore our preliminary
conclusion is that DmGluRA-PI3K pathway inhibition does, indeed, increase perineurial glial
thickness, but also that Push plays an additional role in regulating glial growth. Perhaps Push
regulates perineurial glial growth by acting in the peripheral glia, as well as regulating perineurial
glial growth by regulating excitability from the motor neuron. Resolving this issue is important
but is beyond the scope of the current grant.
Dasgupta B,Yi Y, Chen DY, Weber JD, Gutmann DH (2005) Proteomic analysis reveals
hyperactivation of the mammalian target of rapamycin pathway in neurofibromatosis 1-
associated human and mouse brain tumors. Cancer Res 65:2755-2760.
Howlett E, Lin C-J, Lavery W, Stern, M (2008) A PI3K-mediated negative feedback regulates
neuronal excitability, PLoS Genetics 4: el000277.
Johannessen CM, Reczek EE, James MF, Brems H, Legius E, CichowskI K (2005) The NF1
tumor suppressor critically regulates TSC2 and mTOR. Proc Natl Acad Sci USA 102:8573-
Lavery W, Hall V, Yager JC, Rottgers A, Wells MC, Stern M (2007) Phosphatidylinositol 3
kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral
nerves, J. Neurosci, 27: 279-288.
Yager J, Richards S, Hekmat-Scafe D, Hurd D, Sunderesan V, Caprette D, Saxton W, Carlson J,
Stem M (2001) Control of Drosophila perineural growth by two interacting neurotransmitter-
mediated signaling pathways, Proc. Natl. Acad. Sci., 98: 10445-10450.
Dept, of Biochemistry MS-140
Houston, TX 77251-1892
FAX: (713) 348-5154