Basic HTML Version
Poster Full Abstracts - Drosophila Models of Human Diseases
Poster board number is above title. The first author is the presenter
238
have to be strictly controlled to ensure cellular viability. We study the roles of nuclear receptor DHR96 (
Drosophila
Hormone Receptor 96) in the regulation
of cellular cholesterol homeostasis and metabolism.
DHR96
mutants are viable and display no obvious phenotypes when reared on a normal fly food
medium, but arrest development when reared on a low-cholesterol diet. DHR96 binds cholesterol in vivo, suggesting that cholesterol or a related molecule
acts as a ligand for this receptor. We are now employing genome-wide strategies to identify and validate direct targets of this transcription factor. One
strategy utilizes a transgenic line expressing DHR96 fused to the VP16 activation domain, which allows us to screen for highly activated gene targets via
microarray analysis. We found several Niemann-Pick disease type C2 (
NPC2
) genes, which encode midgut-specific cholesterol transporters, to be likely
direct targets of DHR96. We then examined whether misregulation of
NPC2
genes is the cause for the lethality of
DHR96
mutants on low cholesterol media.
We found that ubiquitous knockdown of one of the
NPC2
genes causes lethality specifically on low-cholesterol media, which is rescuable by cholesterol
supplementation. Consistent with this observation, knocking down
DHR96
specifically in the midgut is sufficient to recapitulate the cholesterol
hypersensitivity phenotype of
DHR96
mutants. We are now conducting genetic epistasis studies to examine whether
NPC2
gene function is required for
DHR96-mediated responses to changing levels of cholesterol. Our studies demonstrate that
DHR96
resides at the top of a genetic hierarchy controlling
cholesterol homeostasis, allowing us to dissect the complex regulatory pathways that maintain a healthy cholesterol balance in all cells.
392B
The Role of
slowpoke
Encoded BK Channel in Heart Function.
Santiago Pineda
1
, Karen Ocorr
1
, Diane Fatkin
2
, Rolf Bodmer
1
. 1) Sanford Burnham
Medical Research Institute, La Jolla, CA; 2) Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010.
Abnormalities in the ion channels that modify cardiac conduction are a common cause of cardiac arrhythmias. One channel that may have an important
role in heart function is the BK channel encoded by the
slowpoke
gene, a K
+
channel that is both Ca
2+
and voltage gated. Previous studies have identified the
BK channel as important in heart function of larva and adult fly. Yet, it remains unclear whether this effect is due to altered neuronal signaling or cardiac
specific. Heart specific RNAi knockdown of the BK channel in a denervated, semi-intact heart prep, we observed a significantly increased diastolic interval
and heart period (decreased rate); suggesting an effect on cardiac repolarization. In agreement with this pharmacological activation of the channel resulted in
decreased heart period (increased heart rate). Yet, the mechanism of BK channel function in the heart is still unknown. We discovered an association
between non-synonymous variants in this channel gene and heart arrhythmias in humans and have expressed both the mutated and wild type human BK
channel gene in flies lacking
Drosophila
BK channel expression. Flies with the mutated channel had poorer heart function compared with controls. We are
using recombineering genetic tools to tag the BK channel in vivo while expressing the channel under control of its native promoter in order to determine
cellular localization. Our preliminary research shows that modification of BK channel activity affects heart function in the fly suggesting that the human BK
mutation is responsible for the arrhythmia seen in the human cohort. Using the fly heart model we should be able to elucidate the specific component of BK
channel function that contributes to these patients’ heart disease.
393C
Modeling reductive stress induced heart disease in flies.
Heng Xie, Kent Golic. Dept Biol, Univ Utah, Salt Lake City, UT.
Alpha-B crystallin (
CryAB
) is a mammalian small heat-shock protein. The R120G amino acid substitution mutation of
CryAB
(
CryAB
R120G
) is associated
with multiple diseases, including cataracts and cardiomyopathy
[1]
. Heart specific expression of
CryAB
R120G
in the mouse resulted in cardiomyopathy,
including cardiac hypertrophy, progressive heart failure and premature death. Hearts expressing
CryAB
R120G
had an excess of reducing equivalents in cells,
and mutation of
G6PD
, one of the primary sources of NADP reduction to NADPH, suppressed the phenotype
[2]
. These results led to the conclusion that
CryAB
R120G
cardiomyopathy results from reductive stress, defined as an excess of reducing equivalents in the cell. To investigate the cellular mechanism of
reductive stress pathology, we expressed the human
CryAB
R120G
gene in flies. Expression in the eye results in an obvious rough eye phenotype. We found
that the eye phenotype was suppressed by mutation of G6PD, and enhanced by overexpression of G6PD, indicating that the basis of cellular dysfunction is
similar in mouse heart and fly eye. We found that this phenotype is connected with NADPH levels rather than the G6PD protein itself, because reduction of
other NADPH producing enzymes (phosphogluconate dehydrogenase (6PGD), malic enzyme (MEN) and isocitrate dehydrogenase (IDH)) also suppressed
the phenotype. Our results extend the mouse experiments to show that all major sources of NADPH affect the
CryAB
R120G
phenotype, strongly supporting the
reductive stress hypothesis. [1] Vicart, P., Caron, A., Guicheney, P., Li, Z., Prévost, M.C., Faure, A., Chateau, D., Chapon, F., Tomé, F., Dupret, J.M.,
Paulin, D., Fardeau, M., 1998 A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat. Genet. 20(1):92-5. [2]
Rajasekaran NS, Connell P, Christians ES, Yan LJ, Taylor RP, Orosz A, Zhang XQ, Stevenson TJ, Peshock RM, Leopold JA, Barry WH, Loscalzo J,
Odelberg SJ, Benjamin IJ., 2007 Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice. Cell
130(3): 427-39.
394A
The inwardly rectifying potassium channel, Irk2, contributes to development of the adult wing in
Drosophila
.
Emily A. Bates
1
, Giri Dahal
1
, Joel
Rawson
2
, Brandon Gassaway
1
, Ben Kwok
1
, Emily Bates
1
. 1) Chemistry and Biochemistry, Brigham Young University, Provo, UT; 2) UT Health Science
Center, San Antonio, TX.
There is currently no defined role for potassium channels in developmental signaling. However, mutations that disrupt function of a human inwardly
rectifying potassium channel, Kir2.1, are associated the morphological defects of Andersen-Tawil Syndrome: cleft palate, incomplete dentition, skeletal
fusion and abnormal curvature of digits. We use
Drosophila melanogaster
to determine how inwardly rectifying potassium channels affect development. In
Drosophila
, the Irk2 inwardly rectifying potassium channel is a homolog to Kir2.1.
irk2
deficient lines,
irk2
RNAi, and expression of a dominant negative
Irk2 subunit demonstrate that Irk2 function is necessary for pattern formation in the development of the adult wing. Compromised Irk2 function causes
wing-patterning defects similar to those found when Decapentaplegic (Dpp) signaling is disrupted. To determine if Irk2 plays a role in the Dpp pathway, we
generated animals that were deficient in both Irk2 and in Dpp signaling. Irk2 dominant negative phenotypes are enhanced by reduced Dpp signal. In
wildtype animals, the Dpp signal can be detected in a stripe along the anterior/posterior boundary of the larval wing imaginal disc. The Dpp signal is reduced
in irk2 deficient animals. Expression of dominant negative Irk2 completely eliminates the Dpp signal in the larval wing disc. TUNEL staining reveals that
expression of a dominant negative
irk2
causes cells in the wing disc to die via apoptosis. Wing phenotypes could be explained by disruption of the Dpp
signal or aberrant apoptosis. Blocking apoptosis with overexpression of P35 in animals that also express the dominant negative
irk2
reveals that apoptosis