Worm Breeder's Gazette 15(1): 42 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
| 1 | Neurogenetics Department, Mass. General Hospital, Charlestown, MA 02129 |
| 2 | Cancer Center, Mass. General Hospital, Charlestown, MA 02129 |
There are striking similarities between vertebrate and C.
elegans nervous systems, both at the cellular and molecular level
lending this organism to the study of human diseases. We have
developed a C. elegans model for Huntington!s disease (HD) and other
disorders that are caused by the expansion of a CAG repeat, coding for
a polyglutamine domain. The repeat is located in exon 1 of the HD
gene, and is polymorphic with normal individuals having 10-34 repeats
and affected individuals having greater that 36 repeats. This mutation
places HD in a (still growing) group of diseases including spinal and
bulbar muscular atrophy (SBMA) and a variety of spinal and cerebellar
ataxia!s (SCA!s). Individuals with HD suffer progressive
neurodegeneration which causes chorea, dementia and, eventually, death.
HD is inherited in an autosomal dominant fashion and phenotypic data
from three independent mouse knockouts suggest that the expanded
CAG/polyglutamine domain results in a gain of function causing neuronal
cell death. Huntingtin is a widely expressed 3144 a.a. novel protein
of unknown function, with the N-terminal polyglutamine domain (the
cloned allele has 23 repeats) followed by a proline rich region, HEAT
repeats and apopain cleavage sites. Although huntingtin levels are
highest in the brain, the affected neurons do not have the strongest
expression levels. The biochemical process by which the expanded
CAG/polyglutamine domain causes specific neuronal cell death in HD (and
related diseases) is poorly understood. Transgenic mice expressing exon
1 of the human huntingtin gene containing over 100 glutamine codons
display a progressive neurologic phenotype and nuclear inclusion
bodies, but fail to show neuronal cell death. There is, in fact, no
current animal or cellular model for HD that features cell death.
Introduction of an N-terminal fragment of the human huntingtin
gene containing an expanded CAG repeat causes neurodegeneration in C.
elegans. In lieu of a C. elegans homolog, we used the osm-10 promoter
to express the first 170 a.a. of normal and mutant (expanded)
huntingtin. osm-10 is expressed in the ASH, ASI, PHA and PHB sensory
neurons of C. elegans (Hart and Kaplan, in prep.). In our first set of
experiments, ASH neuron survival was assayed by staining with DiO, a
fluorescent dye that can be taken up by eight classes of neurons
(staining is dependent on intact sensory processes). Based on DiO
staining, wild-type and transgenic worms containing normal huntingtin
had intact ASH neurons. However, in animals containing the mutant
huntingtin, the ASH neurons failed to stain up to 40% of the time
suggesting degeneration of the sensory processes or cell death. In a
second set of experiments, we used osm-10::GFP to assay ASH survival
and stained the cells with DiI (similar to DiO but on the rhodamine
channel). After looking at five independent arrays, we found that
animals transgenic for the mutant huntingtin had up to 39% ASH neurons
that both failed to stain with DiI and were not detectable with the GFP
fusion protein. These data suggest that the presence of an expanded
CAG/polyglutamine domain within a fragment of the human huntingtin gene
causes cell death in C. elegans. We are currently working to prove that
the neurons are dying, to increase the rate of cell death by improving
expression of the huntingtin constructs and toward integrating the
arrays to eliminate mosiacism. In addition, we will determine if
previously characterized C. elegans cell death pathways are required
for the CAG/polyglutamine neurodegeneration/death. Currently, we plan
to screen for suppressers of this effect and begin to define the
process by which the expanded CAG/polyglutamine domain in HD and other
CAG repeat diseases causes cell death.