Localization and function of the Drosophila huntingtin protein
by
James D. Mediatore
B.A. Biochemistry
University of Colorado at Boulder, 2003
SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN BIOLOGY
AT THE
MASSACHUSETS INSTITUE OF TECHNOLOGY
SEPTEMBER 3, 2007
The author hereby grants to MIT permission to reproduce and to distribute
publicly paper and electronic copies of this thesis document in whole or in part.
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Author:
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A
Department of Biology
2007
. .•1,
Certified by:
J. Troy Littleton
Associate Professor of Biology
Thesis Supervisor
Accepted by
Stephen P. Bell
Professor of Biology
Chairman of the Graduate Committee
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Localization and function of the Drosophila huntingtin protein
by
James D. Mediatore
Submitted to the Department of Biology on September 3, 2007
in partial fulfillment of the requirements for the degree of
Master of Science in Biology
ABSTRACT
Huntington's Disease (HD) is an autosomal dominant neurodegenerative
disorder caused by an expansion of a polyglutamine tract in the huntingtin
protein. This mutation leads to conformational instability, resulting in huntingtin
aggregation and degeneration of neurons in the striatum and cortex. HD is
characterized by motor dysfunction, personality changes, dementia, and early
death. Although a number of abnormal cellular phenomena have been described
in systems modeling HD, the specific events initiating pathology remain unclear.
It is widely viewed that inclusions may have a toxic gain-of-function which is
central to HD pathogenesis. However, evidence is accumulating that supports
the loss of huntingtin function as a likely contributor to the unravelling of cellular
processes early in the course of the disease.
The fruitfly Drosophila melanogaster has an orthologous huntingtin gene
with several regions showing 40-50% similarity to mammalian huntingtin at the
amino acid level. Like the mammalian huntingtin gene, the fly huntingtin lacks
sequence motifs that would suggest functional correlates to other known
proteins.
I have pursued a cell biological and physiological analysis of Drosophila
melanogastermutants with reduced huntingtin gene expression. Normal levels of
huntingtin were not required for normal localization of mitochondria in neurons,
synaptic transmission in the visual system, or formation of synapses.
Thesis Supervisor: J. Troy Littleton
Title: Associate Professor of Biology
TABLE OF CONTENTS
Title Page................................................................................1
Abstract...............
2......
............................................................
Table of Contents.................................................................3.......
Chapter I:Introduction...........................................................................4
Chapter II: Results...........................................................................19
Materials and methods............................... .............
30
Chapter III: Concluding remarks............... .......................................
31
Appendix A: Genetic schemes................................................33
References.......................................................34
Chapter I:Introduction
Huntington's Disease: Background
Huntington's Disease (HD) is the most common inherited late-onset
neurodegenerative disease, which afflicts approximately 1 in 10,000 individuals
in North America and Europe (HDCRG, 1993). HD is a progressive disorder
typically commencing in middle age (mean age of 35). However symptoms have
been observed in extreme cases in infants and in adults over 80 (Myers, 2004).
HD is characterized by involuntary choreiform movements, loss of motor
coordination, dementia, personality changes, and ultimately premature death
within 10-15 years following onset of symptoms (Vonsattel et al., 1985).
HD is caused by an expansion in the polyglutamine (polyQ)-encoding
'CAG' nucleotide repeat domain in the 5' region of the gene, which resides at
4p16 in the human genome (HDCRG, 1993). The HD phenotype is exhibited
when the expansion of this tract occurs beyond a threshold number (-35)
(Rubinsztein et al., 1996). Age of onset and severity of symptoms are correlated
with length of the CAG expansion, with longer repeats resulting in earlier age of
onset and more rapid progression of symptoms, culminating in mortality (Duyao
et al., 1993). Pathogenic repeat lengths exhibit the phenomenon of 'anticipation',
where the repeat length is unstable and tends to increase in size when
transmitted to subsequent generations (Mclnnis, et al. 1996). There is currently
no cure or effective therapy for HD.
1;
0bp
S
-
I
nITs
(CAG)n
-3T
Huntington's disese gne
(O)n
35AkDa
Huntingtin
o60
IS20
ii
-I
2O
I
..
4be CAG
0of
Number
eL
w
mI
of CAG Repeat Units
m
I
120
FIGURE 1. Polyglutamine length-dependence of HD age of onset. Schematic
showing the relative location and variable length of the polyglutamine domain of
the human huntingtin gene. Repeat number is inversely corellated to age of
onset. (Gladstone Center for Translational Research, University of California at
San Franscisco.)
HD is among a family of late-onset, progressive neurodegenerative
polyglutamine diseases showing an autosomal dominant pattern of inheritance
(see Figure 2). Common to all of these diseases is polyQ expansion and
subsequent conformational instability, leading to the accumulation of abnormal
forms of the protein. In each condition the pathogenic protein is expressed
ubiquitously in the brain, however the neuronal subtypes exhibit a selective
vulnerability leading to characteristic neurodegenerative symptoms specific to
each disorder. This phenomenon is observed more generally in other
neurodegenerative diseases involving protein misfolding, including Alzheimer's
disease, amyotropic lateral sclerosis (Lou Gherig's disease), Parkinson's
disease, and the spongeiform encephalopathies (prion diseases). In general the
onset of symptoms is loosely correllated with the formation of neuronal
inclusions. In HD, huntingtin-immunopositive inclusions are present in neurons
that degenerate, however the presence of such inclusions does not exactly
correspond with neurodegeneration (Vonsattel et al., 1985; Kuemmerle et al.,
1999). The role of huntingtin aggregation in HD pathogenesis continues to be
controversial and specific pathways leading from protein misfolding to
pathogenesis remain obscure.
HD brains show widespread abnormalities including dystrophic neurites
and apoptotic neuronal death, displayed most prominently in the striatum and
cortex, resulting in a loss of brain weight of up to 30% (Aylward et al., 1997).
Neuropathology in HD patients is highly selective, with substantial loss of
neurons in the caudate and putamen of the basal ganglia, particularly GABAergic
type II medium spiny neurons, which comprise 80% of striatal neurons (DiFiglia
et al., 1991). These neurons receive glutamatergic signals from the cerebral
cortex and are involved in motor control, the loss of which is consistent with HD's
characteristic choreiform disorder (Albin et al., 1990).
Normal
Disease Gene locus Gene product CAG(n)
Androgen
SBMA Xq11-12 receptor
9-36
HD
4p16.3
Huntingtin
6-34
SCA1
6p22-23
Ataxin-1
6-44
39-82
Protein
localization
Nuclear and
cytoplasmic
Cytoplasmic
Nuclear in
neurons
SCA2
12q23-24 Alexin-2
15-31
36-63
Cytoplasmic
SCA3
14q24.3-31 Ataxin-3
12-41
2-84
Cytoplasmic
SCA6 19p13
CACNAIA
Expended
CAG(n)
38-62
36-121
Special features
Brain regions most affected
Anterihorn and bulbar neurons,
dorsal root ganglia
Intermediate alleles: 29-35
Striatum, cerebral cortex
Normal alleles >21 repeats
Cerebellar Purkinje cels, dentate
interrupted with 1-4 CAT units nucleus; brainstem
Normal alleles interrupted with Cerebellar Purkinje cells, brain stem,
1-2 CAA units
fronto-temporal lobes
Cerebellar dentate neurons, basal
ganglia, brain stem, spinal cord
Cerebellar Purkinje cells, dentate
Cal membrane
4-18
1-33
4-35
37-306
Nuclear
6-36
49-84
Cytoplasmic
nucleus, inferior olive
Cerebellum, brain stem, macula,
SCA7
3p12-p21.1 Ataxin-7
DRPLA 12q
Atrophin-1
Intermediate alleles: 28-35
visual cortex
Cerebellum, cerebral cortex, basal
ganglia, Luys body
FIGURE 2. Polyglutamine diseases. Spinobulbar muscular atrophy (SBMA),
Huntington's disease (HD), and the spinocerebellar ataxias [including
dentatorubropallidoluysian atrophy (DRPLA)] are the 8 neurodegenerative diseases
caused by polyglutamine expansion. All are dominantly inherited, with the exception of
SBMA. Although the basis of molecular dysfunction is common to all, the symptoms and
regions of brain pathology are distinct. (Reproduced from Zoghbi et al., 2000)
Huntingtin form and potential function
The precise function of the huntingtin gene is unknown. Determining the
early pathogenic events in Huntington's Disease and distinguishing them from
downstream effects is essential to understanding the disease and developing
essential treatment strategies. The huntingtin protein is a very large (348kD),
soluble protein expressed ubiquitously and enriched in the brain and testes
(Difiglia et al., 1995). Human huntingtin comprises 3,144 amino acids and largely
lacks protein domains with defined biological function, thus frustrating efforts to
situate it within the family of other proteins with well-defined biological roles.
antiparallel
N
cV-
parallel
C
c
c
C
compact
random
coil
c
N
Zc
hairpin
compact
C 1-sheet
N
P-helix
FIGURE 3. P-sheet models of polyglutamine aggregation. In this schematic
representation of several proposed structural models of misfolded polyQ
domains, 1-sheet is represented by the red zig-zag lines. (a) The extended
antiparallel 1-sheet, or "polar zipper" model (Perutz et al., 1994), or alternatively
a parallel P-sheet (b), anti-parallel 1-hairpin (c), compact random coil composed
of four anti-parallel (d)or 1-strand (e) elements, and finally (f) a regular parallel 1helix with periodicity of 20 residues. (Reproduced from Ross et al., 2003)
Huntingtin is associated with various organelles including the nucleus,
golgi apparatus, and endoplasmic reticulum, and localizes to synapses where
huntingtin associates with clathrin-coated vesicles, endosomal vesicles, and
microtubules (Velier et al., 1998; Hoffner et al., 2002; Kegel et al., 2002). This
widespread subcellular localization and the inability to find structural and
sequence homology with other known protein domains frustrated initial efforts to
define huntingtin cell function.
The polyglutamine repeat region is often modeled to form a polar zipper
structure, which may be involved in associations with Q-rich domains in
transcription factors, including cAMP-responsive-element binding protein (CBP),
p53 (McCampbell et al., 2000; Steffan et al., 2000), Spl, TAFII11130 (Dunah et al.,
2002), N-CoR, and Sin3A (Boutell et al., 1999). Huntingtin's polyproline (polyP)
region has been implicated in interactions with dynamin, huntingtin-interacting
protein 1 (HIP1), and SH3-containing Grb2-like protein (SH3GL3) (Qin et al.,
2004). Interactions through this polyP region may be direct associations with SH3
and WW protein domains, or mediated by structural stabilization of the
polyglutamine domain. Other huntingtin-interacting proteins are huntingtinassociated protein 1 (HAP1), which binds with the pl50glued subunit of dynactin
(Li et al., 1998). Both huntingtin and HAP1 are transported bidirectionally in
neurons (Block-Galarza et al., 1997).
Other interacting partners are protein
kinase C and casein kinase substrate in neurons 1, postsynaptic density-95, and
FIP-2 (Harjes and Wanker, 2003). These proteins are involved in vesicle
transport,
clathrin-mediated
endocytosis,
apoptosis,
cell-signalling,
morphogenesis, and transcriptional regulation, which suggests that huntingtin
may have a role in several diverse processes.
SUMO/UBIL
"AA
,_ 1_©
0
250
600
750
1,000 1,250
1,500 1,750 2,000 2,250 2,500 2,750
3,000
FIGURE 4. Schematic of huntingtin's amino acid structure. (Q)n and P(n) represent
the polyglutamine and polyproline regions, respectively. The 37 HEAT repeat domains
are clustered into 3 main groups (red boxes). The circles indicate locations of posttranslational modifications (sumoylation/ubiquitination at the red circles, phosphorylation
at blue). Arrowheads and triangles indicate sites of caspase and calpain cleavage,
respectively. NES isthe nuclear export signal sequence. (figure from Tartari et al., 2005)
Huntingtin has an active C-terminal nuclear export signal and a partially
active nuclear localization sequence, which suggests huntingtin may be involved
in transporting proteins between the nucleus and cytoplasm (Xia et al., 2003).
This is consistent with huntingtin's nuclear and perinuclear localization. The
deletion of the domain involved in associating with the nuclear protein TPR
results in nuclear accumulation of huntingtin (Cornett et al., 2005).
Downstream of the polyglutamine region are the so-called 40-amino acid
HEAT repeat domains, which occur repeatedly in Huntingtin, Elongation factor 3,
protein phosphatsase 2A, and TOR1 proteins, and are thought to be involved in
protein-protein interactions (Andrade and Bork, 1995). Human huntingtin has 37
putative HEAT repeat domains in 3 clusters. Drosophila huntingtin has 28 HEAT
repeat domains (Takano et al., 2002). The presence of these domains indicate
that huntingtin could play a role as a molecular scaffold or adaptor for a variety of
different cargos undergoing cellular trafficking.
Four types of post-translational modification of huntingtin have been
described. The N-terminal lysines are involved in sumoylation, which reduces the
ability of N-terminal fragments to form aggregates (Steffan et al., 2004), and
ubiquitination (Kalchman et al., 1996). The phosphorylation of serines at
positions 421 and 434 in the human protein are associated with proteolytic
cleavage. Phosphorylation of these residues is reduced in the disease form of
the protein and is associated with increased cleavage and toxicity (Luo et al.,
2005). Huntingtin is palmitoylated by huntingtin-interacting protein 14, which is
necessary for its trafficking in axons and also consistent with its proposed role in
vesicular trafficking (Difiglia et al., 1995; Yanai et al., 2006).
Protein localization studies are consistent with a role for endogenous
huntingtin in axonal transport. Immunohistochemical studies in mammalian
systems reveal huntingtin labeling in the neuronal cytoplasm, nerve tracks,
intense punctate staining at synapses, and around secretory vesicles at the
ultrastructural level (DiFiglia et al., 1995). Normal huntingtin undergoes fast
axonal transport in both anterograde and retrograde directions (Block-Galarza et
al., 1997). Neurodegeneration could result from interruption of trafficking of
essential neurotrophic factors, e.g. BDNF, by mutant huntingtin. In accordance
with this hypothesis, studies of Alzheimer's disease and other non-polyQ
diseases have found that neurodegeneration is a consequence of defective
axonal transport.
This variety of potential activities suggest diverse cellular roles for
huntingtin. It is conceivable that the huntingtin protein is flexible in form
depending on its interacting partners, lending it multi-functionality according to
the timing and arrangement of its subcellular localization. This hypothesis is
supported by the evidence that different huntingtin epitopes are reactive to
different antibodies depending on the subcellular localization of the protein (Ko et
al., 2001).
Mechanisms of pathogenesis
The causative mutation in HD is an expansion of glutamine-encoding CAG
triplet repeats in exon 1 of the HD gene, huntingtin, beyond a threshold of 35-40.
Huntingtin is a very large (3,144 amino acid, -350kD) protein expressed not only
in the brain of humans and mice, but also in testes, liver, heart, lungs, and
pancreatic islets as well (Ferrante et al., 1997; Cattaneo et al., 2005).
A central point of debate in HD research is the role of huntingtinimmunopositive intracellular aggregates. Evidence has supported diverse claims:
that they instigate neurodegeneration (Yang et al., 2002), are merely by-products
of the neurodegeneration process (Szebenyi et al., 2003; Saudou et al., 1998), or
are even neuroprotective in nature (Arrasate et al., 2004). Aggregate formation
has been shown by several lines of evidence to be promoted by truncated polyQexpanded N-terminal huntingtin fragments. A consensus is emerging that a
cascade of cytopathic events culminate in the HD phenotype.
Caspase cleavage
Huntingtin contains 3 well-characterized protease cleavage sites, which
are involved in the fragmentation of both wild-type and mutant huntingtin,
although the mutant form is more susceptible to cleavage and fragments
accumulate in the nucleus and cytoplasm (Goldberg et al., 1996; Wellington et
al., 1998; Gafni et al., 2004). Caspase cleavage sites are conserved in
vertebrates but lacking in Drosophila. The role of proteolysis in huntingtin
function is not understood. However, experiments inhibiting caspase and calpain
activity in cells reduces cleavage of the mutant protein and reduces toxicity
(Wellington et al., 2000; Gafni et al., 2004).
Proteolytic cleavage produces short, toxic N-terminal fragments containing
the polyQ expansion. Expression of huntingtin exon 1 containing a pathogenic
polyQ tract is sufficient to produce HD-like symptoms in mice and flies. These
truncated fragments have been demonstrated to form intracellular aggregates
more easily compared to polyQ expansions in the context of longer proteins.
Recently investigators have shown that specific Cdk5 phosphorylation of
huntingtin in cultured human neurons reduces huntingtin cleavage, and
susbsequent toxicity and aggregate formation, while products of cleavage
specifically inhibit the use of huntingtin by Cdk5 as a substrate. This suggests a
scenario wherein toxic huntingtin fragments compromise the ability of Cdk5 to
restrict huntingtin cleavage, resulting in a positive feedback loop and rapid
accumulation of pathogenic huntingtin cleavage products. Soluble mutant
huntingtin has also been observed to interact with mTOR kinase leading to its
loss of function, suggesting that mutant huntingtin may bind to several kinases
and cause diverse physiological changes in cells. The ability of Drosophila
huntingtin to function as a substrate for proteolysis in flies has yet to be
determined.
Transcriptional dysregulation and excitotoxicity
Both normal and mutant forms of huntingtin have been shown to interact
with a host of transcription factors, including TAFII11130 and Spl (Dunah et al.,
2002), and the cAMP response element binding protein binding protein (CBP)
(McCampbell et al., 2000). Normal huntingtin stimulates levels of cortical BDNF
by acting at the level of BDNF gene transcription, an effect which is lost in the
presence of the mutant protein (Zuccato et al., 2001). Cleavage of full-length
huntingtin has been shown to yield toxic huntingtin N-terminal fragments in mice
which localize to the nucleus and sequester Q-rich transcription factors in
aggregates. These reports support a general model of transcriptional
dysregulation as a significant contributing factor of HD pathology. This could
arise from loss of normal interactions between huntingtin and transcription factors
or abnormal interactions arising from polyglutamine expansion in the pathogenic
form.
Medium spiny neurons (MSNs) in the striatum are most severly affected in
HD. MSNs are innervated by glutamatergic axons, glutamate being the primary
excitatory neurotransmitter in the mammalian brain. This observation led to the
hypothesis that mutant huntingtin exerts an excitotoxic effect on these neurons,
and has found support in experiments wherein selective loss of MSNs and HDlike symptoms are produced when glutamate receptor agonists are delivered to
the striatum of test animals.
Huntingtin loss of function phenotypes
Huntingtin is critical for early embryonic development in mice, as
homozygosity for inactivated huntingtin alleles generated by targeted disruption
results in death around embryonic day 8, before the nervous system is formed.
Null embryos form the three germ layers, but these become disorganized and
malformed during gastrulation. Mice expressing less than 50% of normal
huntingtin levels are viable, however they show abnormal brain development,
neurodegeneration, and sterility (White et al., 1997; Auerbach, et al. 2001; Nasir
et al., 1995). Mice that develop in the presence of huntingtin but lose normal
huntingtin in post-mitotic neurons show an HD-like phenotype (Dragatsis, et al.,
2000).
Reduced levels of huntingtin lead to abnormal distribution and morphology
of cellular organelles in murine embryonic stem cells (Hilditch-Maguire et al.,
2000). Transport of vesicles and mitochondria is diminished in Drosophila
expressing siRNAs targeting huntingtin (Gunawardena et al., 2003) and in
neurons from conditional huntingtin -/- mice (Trushina et al., 2004). Furthermore,
decreased huntingtin expression in flies with only 50% of genetic complement of
the motor proteins kinesin or dynein exhibited axonal swellings and organelle
jams characteristic of flies with severe axonal transport deficits due to 100% loss
of these motor proteins (Gunawardena et al., 2003; Pilling et al., 2006). This
suggests that huntingtin interacts with these motor proteins at some level in the
process of organelle transport, and that it is plausible that huntingtin loss-offunction may contribute to axonal swellings and blockages in HD models where
the pathogenic protein is expressed. In the context of HD, a huntingtin loss of
function model is supported by the observation that wild type huntingtin is
sequestered among misfolded mutant huntingtin proteins within cytoplasmic
aggregates.
Axonal transport defects
More recently, several reports have revealed a new mechanism by which
mutant huntingtin disturbs brain function: disruption of axonal transport. This
defect could be a manifestation of axonal blockage by huntingtin aggregates, or
by a loss of function phenotype.
Evidence suggesting axonal blockages are formed from huntingtin
aggregates has come from studies in cultured neurons and in Drosophila (Li et
al. 2001; Lee et al., 2004). Huntingtin has been reported to be involved in axonal
trafficking in rat sciatic nerve (Block-Galarza, J., et al. 1997), while reduced
amounts of huntingtin interfere with proper axonal transport in larval neurons in
Drosophila (Gunawardena et al., 2003). Polyglutamine-dependent inhibition of
retrograde and anterograde transport has also been documented in squid giant
axons (Szebenyi et al., 2003). These findings are consistent with other studies
that
show
polyglutamine-dependent
axonal
pathology
precedes
neurodegeneration in C. elegans and mice (Parker et al., 2001; Li et al., 2001).
Mitochondrial toxicity
Mitochondria are normally trafficked bidirectionally in neurons. Healthy
mitochondria that produce ATP and are needed to maintain membrane potential
and calcium homeostasis are moved to the synapse, while damaged, aged
mitochondria are moved to the cell body for repair or disposal in lysosomes
(Miller and Sheetz, 2004). Deficiencies in normal mitochondrial localization may
contribute to a retarded ability to respond to metabolic buffering needs, leading to
neuronal dysfunction, while persistence of damaged mitochondria could lead to
generation of reactive oxygen species that cause cellular damage and apoptosis
(Lee and Wei, 2000). Transport of vesicles and mitochondria is diminished in
Drosophila (Gunawardena et al., 2003) and in neurons from conditional
huntingtin -/- mice (Trushina et al., 2004).
Nucle
Mutant
gene
C
os
PrOtoego~me
CyoLysosomeparn
Cytaplamr
FIGURE 5. Potential pathways of polyglutamine pathology. (Adapted from Rudnicki
and Margolis, 2003) (a)The pathogenic process (blue arrows) begins with the synthesis
of a protein with an expanded polyglutamine (polyQ) tract. (b) The expanded
polyglutamine tract alters the native conformation of the protein, which is reinforced by
the presense of molecular chaperones. Misfolded protein undergoes two distinct
proteolytic processes: (c) lysosomal-dependent proteolysis; (d) some protein is
ubiquitinated (Ub) and degraded via the proteasome. (e) Cleavage produces an Nterminal fragment that is prone to aggregation. (f) The mutant proteins proceed from a
monomeric random coil or O-sheet into oligomeric n-sheets and eventually into insoluble
aggregates. (g)These species undergo abnormal interactions with cellular proteins, or in
another model might represent a mechanism for reducing the toxicity of aggregation
intermediates by sequestering toxic monomeric forms of the mutant protein. (h)
Aggregation intermediates inhibit proteasomal processing. (i) The monomers or
oligomers directly activate caspases or disrupt mitochondrial function. (j) Aggregates
translocate into the nucleus (by an unknown mechanism) and (k)recruit specific nuclear
factors, co-activators and co-repressors, inhibiting their normal activities and (I) resulting
in altered gene transcription.
Chapter II: Results
Characterization of huntingtin function in Drosophila
Abstract
To observe how a loss of huntingtin function may contribute to abnormal
trafficking of mitochondria in motor neurons, I examined the distribution of a
GFP-tagged mitochondrial protein in motor neurons of live Drosophila under
conditions of reduced huntingtin gene expression. There was no obvious
difference in mitochondrial localization in the motor neurons of 3 d instar larvae
expressing a transgenic dsRNA hairpin reported to significantly reduce huntingtin
gene expression via RNA interference or in embryos injected with siRNAs.
In order to observe the localization of endogenous huntingtin protein in
Drosophila and evaluate the extent of gene expression in flies expressing
reduced levels of huntingtin protein, I pursued the generation of antibodies
specific to Drosophila huntingtin. Unfortunately these antibodies were not
reactive to endogenous fly huntingtin via Western or immunological approaches.
To evaluate how loss of huntingtin function affects neuronal survival and
physiology in Drosophila, I pursued strategies to create a huntingtin null mutant. I
examined retinal neuron function and resistance to stress via electroretinograms
(ERGs) in huntingtin null flies. There was no significant difference between ERGs
in huntingtin null flies and controls.
Introduction
In spite extensive efforts to understand the basis of HD pathology from the
perspective of mutant protein misfolding and aggregation, the body of information
resulting from these studies is often confusing. However recent evidence
suggests HD pathology may be in part due to a loss of function of wild-type
huntingtin. Models positing protein aggregation and wild-type loss of function are
not mutually exclusive however, as wild-type huntingtin is sequestered in
aggregates inside cells expressing the mutant protein.
The Drosophila huntingtin (Htt) gene is 11 kB and encodes a large 396 kD
protein that, like its vertebrate homologs, lacks domains that are conserved
among known protein families. The fly ortholog lacks the continuous
polyglutamine stretch that characterizes the mammalian Htts, although it retains
three large regions showing approximately 25% amino acid sequence identity
and 50% similarity. The dHtt locus is comprised of 29 exons and spans 43kB of
genomic DNA at cytologic band 98E on the 3rd chromosome. Like the
mammalian gene, the fly version lacks sequence motifs that would suggest
functional correlates to other known proteins (Li et al., 1999). Vertebrate
huntingtin sequences are highly conserved, with human and zebrafish
sequences sharing 70% identity at the amino acid level. In contrast the
Drosophila sequence is relatively dissimilar to those of vertebrates. The fly
homolog lacks the polyglutamine repeat domain and the adjacent polyproline
domain characteristic of human huntingtin (Li et al., 1999). The fly homolog is the
largest of the huntingtin family, extending several hundred amino acids beyond
other vertebrate huntingtin sequences. The transcript is widely-expressed at low
levels throughout the developmental cycle of Drosophila (Li et al., 1999).
Initial models of HD in flies were created by injecting flies with glutamine
expansions and subsequently observing a neurodegenerative phenotype. At
present our HD model system is comprised of transgenic flies expressing the Nterminal region of the human huntingtin gene containing pathogenic or
nonpathogenic numbers of glutamines under control of the UAS/GAL4 binary
expression system. Adult flies expressing pathogenic (Q128) huntingtin in the
nervous system
exhibit abnormal
grooming
behavior, defective motor
coordination, and significantly abbreviated lifespan, while flies expressing nonpathogenic isoforms (QO) are indistinguishable from wild type animals. Larvae
expressing huntingtin-Q128 display cytoplasmic aggregates throughout the CNS,
including axonal blockages that trap synaptic proteins. These blockages are
absent in animals expressing non-pathogenic huntingtin-Q0, Q127 alone, or
Q108 in the context of the non-pathogenic dishevelled gene (Gunawardena et
al., 2003).
Results
Characterization of the cellular localization of the Drosophila huntingtin protein.
Polyclonal antibodies were purified from an exisiting stock of antisera
raised against the N-terminal 319 amino acids of the Drosophila huntingtin (dhtt)
protein from 3 different rats (figure 6A). Antibodies were affinity purified in batch
with GST-dhtt N319 bound to glutathione sepharose beads (Amersham
Biosciences). From the three independently derived batches of sera (#188, 189,
and 190), batch #188 showed the most reactivity and least background in probes
of blots of the recombinant immunogen and endogenous protein isolated from fly
heads. Unfortunately, immunostaining with these antibodies failed to reveal any
reactivity to any structures in fixed 3 rd instar larvae.
New polyclonal antibodies were generated in two rabbits against the same
N-terminal region of the protein. This second batch of polyclonal antibodies did
not show consistent reactivity in Western blots with protein from wildtype
Drosophila or immunostaining of 3 rd instar larvae.
To obtain samples for Western analysis of huntingtin protein expression in
Drosophila, flies were frozen in liquid nitrogen and vortexed to isolate 20 heads
of each genotype.
A
PolyQ
N
ll].V I 1111III
Human
11 II
B
#188antiserum
-220 kD
1111[11111 I
313 5
Predcted
HEAT-Like
Motif
C
#188 antibodies
(pur•id)
-
Canton S fly head protein
FIGURE 6. Immunoreactivity of Drosophila huntingtin antibodies. (A) Animals were
injected with recombinant protein corresponding to the N-terminal 319 amino acids of the
fly huntingtin gene (boxed region). (B) Huntingtin antibodies were used at several
concentrations and detected using a goat anti-rabbit antibody conjugated to HRP
(Jackson ImmunoResearch Laboratories). Protein extracts from 5 to 0.5 heads in
sequentially decreasing amounts was loaded ineach lane.
In vivo observations of mitochondrial transportin huntingtin hypomorphs
In order to see if reduced huntingtin gene expression results in
mislocalization or accumulation of mitochondria in neurons, which would be
consistent with a role for huntingtin in axonal trafficking of these organelles, I
observed how GFP-tagged mitochondria moved in real time in individual motor
neurons in 3 rd instar larvae using fluorescent microscopy. I recombined the 3 rd
chromosome P{GawB}D42 GAL4 driver, which drives expression of UAS-tagged
genes in motor neurons of
3 rd
instar larvae, with a
3 rd
chromosome UAS-
mitoGFP insertion to make a homozygous line expressing fluorescently marked
mitochondria in motor neurons (figure 7). Mitochondrial distribution and axonal
23
morphology
in animals
expressing
dsRNAs
against
huntingtin
RNA
(Gunawardena et al., 2003) were indiscernible from control larvae with wild-type
huntingtin. Injection of D42 GAL4; UAS mitoGFP embryos with two different
siRNAs targeting huntingtin likewise did not show any phenotypic deviation from
control animals injected with the antisense oligonucleotides.
Although preliminary observations of neuronal GFP-tagged mitochondria
in two different RNAi systems failed to show any phenotype, I did not confirm that
any reduction in huntingtin RNA or protein was achieved through these methods.
Approximately eighty 21-nucleotide candidate siRNA target sequences
with optimal efficiency were generated using a web utility from Ambion
(http://www.ambion.com/techliblmisc/siRNA_finder.html). From these eighty, two
were selected based on minimal sequence overlap with non-target sequences
that might complicate interpretation of any phenotype due to non-target gene
knockdown. The sequences, corresponding to bases 308-328 and 1265-1285 of
the Drosophila huntingtin mRNA transcript, had an E value of 3.8 for non-target
sequences from a BLASTN search of the Drosophila genome map. siRNA
injection of Drosophila embryos was performed by Zhou Guan. Drosophila
expressing a GFP-tagged mitochondrial protein in D42 motor neurons were used
in the procedure so that mitochondrial trafficking could be observed in
experimental animals with decreased huntingtin expression. There was no
significant phenotype in flies injected with siRNAs targeting huntingtin compared
to control injected flies.
Huntingtin mutagenesis approaches in Drosophila.
In order to study any loss of function huntingtin phenotypes in Drosophila,
I pursued three different approaches to generating a mutant with reduced
huntingtin gene function. Initially a strategy was pursued to generate a huntingtin
mutant by mobilizing the P-element P{GT1}BG01706 out of its site approximately
100kB 5' to the predicted huntingtin promoter and into the gene itself. This Pelement is located approximately 200kB 5' of the huntingtin locus (figure 8a). I
screened several hundred independently-derived lines, however no eye color
changes (indicative of P-element transposition due to position effects of eye color
marker) were observed in the F2 generations.
This approach was abandoned following our discovery of an
alternative library of flies containing a piggyBac transposon inserted in a noncoding sequence within the huntingtin locus. Strain c07030 from the Exilixis
library contains a piggyBac element less than 400bp from the predicted
huntingtin promoter (Figure 8b). piggyBac transposons can tag and disrupt genes
without the insertion biases of P-elements, however piggyBac elements only
excise precisely, therefore mutagenesis via imprecise excision is not possible
(Thibault et al., 2004). These flies were used as a template for mutagenesis of
the huntingtin locus by gamma ray irradiation. Screening of over 1,000 F1
offspring for loss of red eye color due to lesioning of genomic DNA in the vicinity
of the piggyBac produced no potential candidates.
The third and final strategy for pursuing a huntingtin mutant involved
precise excision of DNA between two transposable elements flanking huntingtin
genomic sequence. Within the Exelixis collection of PBac transgenic flies, we
found three strains of Drosophila with piggyBac transposons inserted into noncoding regions near or within the huntingtin locus (Figure 7A)(Thibault et al.
2004). The sequences of these transposon constructs include short (48-bp) FRT
recombination sites from the yeast 2p plasmid. In the presence of FLP
recombinase expressed via an exogenous Hsp70 promoter on the X
chromosome, the intervening huntingtin coding sequence flanked by the PBac
FRT sites is excised precisely and with relatively high efficiency.
FLP recombinase-expressing females bearing each of the dHtt PBac
alleles were mated to white-eyed males with the third chromosome balancer TM3
GFP. I created -200 lines derived from single flies coming from this crossing, the
non-balancer third chromosome in each line being uniquely derived from an
individual transposition event. The mutagenesis plan called for screening for
putative dHtt recombinant F1 progeny via a PCR-based strategy to confirm
deletion of the targeted Htt sequence in the excision event. However at this time
we discovered that a huntingtin mutant had been generated using this strategy
by Sheng Zhang from the Perrimon lab at Harvard Medical School. Subsequently
I pursued phenotypic analysis of these flies.
(A)
24530k
24520k
24510k
Gene Span
CG9990
24540k
24550k
htt
I
II
24560k
--------
Non coding EM
PBac{PB)huntingtin[c070301
Natural transpo•on
Tranagene inwtion site
(GTi3BG01706
PBacPBahuntingtinin[c003893
A
PBacti-huntingtin[fO4684)
A
(B)
FR"
piggyBac WH
tMS
(c)
A
B
C
FIGURE 7. Genomic maps of Drosophila strains containing transposons
used in mutagenesis of huntingtin gene. (A) Schematic showing position of
the P element used in the initial transposition and imprecise excision strategy,
and the three piggyBac elements used in final mutagenesis attempt. (B)
Composition of piggyBac elements in WH strains from Exilixis collection (Thibault
et al. 2004). (C) Recombination resulting in deficiencies is efficient (-10%) when
proximal (<100kB) FRT sites are recombined in trans in the presence of FLP
transposase (Parks et al. 2004).
Analysis of Huntingtin mutant flies
To detect any abnormal synaptic transmission at a time when behavioral
motor defects were evident, I measured electroretinograms (ERGs) in the visual
system of adult flies whose huntingtin gene was deleted. ERGs are extracellular
recordings of photoreceptor depolarization that indicate synaptic transmission to
second order neurons in response to light. Synaptic events occur at the onset
and termination of a light pulse and are represented by the on- and off-transients
of the ERG. Mutants that disrupt synaptic transmission lose on/off transients
(Littleton et al., 1998). Young mutant flies (adults 1-2 days old) show a normal
ERG response at 20 degrees and when stressed at 37 degrees (figure 9A). Both
old (40-45 days) and young flies show inconsistent loss of ERGs: the mutants
show a higher percentage loss of ERGs under these conditions (-60%), however
the control animals show the same loss albeit at a lesser penetrance (20%)
(figure 9B). There was no consistent defect in spiking or seizure activity in the
DLM when assayed at either temperature.
Control
3 days
dhutt '-
I da0
dhtt
"
43 days
2OSU*C
37-C
14
m
P
h.|
ALkH -•kff
ti
Conrml
FIGURE 8. Electrophysiological analysis of Htt mutants by Sudipta Saraswati
and J. Troy Littleton. (A) Electroretinograms (ERGs) recorded from control and
Htt mutants aged 40-45 days at 250 C, or from Htt mutants aged 1-3 days. Flies
were rapidly heated from 200C to 370C, with test light pulses (black bar below
trace) given at regular intervals. Htt mutants showed a more severe
temperature-induced loss of phototransduction than controls, suggesting Htt
mutant photoreceptors were stress-sensitive compared to controls. (B) Percent
of adult animals aged 40-45 days with a loss of phototransduction at 370C. The
number of preparations analyzed was: control (10); Htt (16).
Materials and methods
Antibody generation
Recombinant immunogen was expressed in BL21 E.coli cells as an Nterminal GST fusion in E. coli from the pGEX2T plasmid encoding the N-terminal
319 amino acids of the Drosophila huntingtin gene. Two rabbits were immunized
as per standard protocol (Invitrogen: http://www.invitrogen.com/content.cfm?
pageid= 3987).
Huntingtin mutagenesis in Drosophila
To
mutate
the
huntingtin
gene
with
gamma
rays,
male
piggyBac{PB}huntingtinc07030 Drosophila containing a w' eye color marker
approximately 125 bp from the 5' end of the huntingtin locus were dosed with
4,000 RADs at 662 kEV from a 137Cs source and mated with w/ -virgins.
Electrophysiology in Huntingtin mutants
Electrophysiological analysis of wandering stage 3rd instar larva was
performed in Drosophila HL3.1 saline (NaCI, 70 mM; KCI, 5; MgCI 2, 4; CaCI 2, 0.2;
NaHCO 3, 10; Trehalose, 5; Sucrose, 115; HEPES-NaOH, 5; pH 7.2) using an
Axoclamp 2B amplifer (Axon Instrument). Electroretinograms were performed as
previously described (Rieckhof et al., 2003). Temperature shifts were performed
by heating mounting clay encompassing the fly to the desired temperature with a
peltier heating device.
Chapter III: Concluding remarks
In conclusion, the huntingtin loss of function phenotype appears to be very
subtle in Drosophila and may not be a contributing factor in the gross defects
observed in axonal transport in existing Drosophila HD models. However the
physiological basis of mild movement defects observed in these huntingtin
mutants remains to be determined. An investigation into genetic interactions
between huntingtin and other axonal motor protein genes, such as kynesin,
dynactin, and pl50Glued may help to elucidate the mechanism by which
huntingtin performs any role at synapses and facilitates vesicular and organelle
transport along axons.
It remains to be reconciled how the null Huntingtin mutant we studied fails
to demonstrate a phenotype consistent with UAS-GAL4 dsRNA loss of function
huntingtin flies reported by Gunawardena et al., 2003. A more basic question is
how Drosophila manage to develop and largely achieve full neuronal functions,
which are severely disturbed in mammalian Huntingtin mutants. It would be
interesting to see if Huntingtin null flies have a more exaggerated disease
phenotype when the mutant form of the protein is expressed, in experiments akin
to those showing that wildtype huntingtin mitigates the effects of the pathogenic
form (Ho et al., 2001; Leavitt et al., 2001).
In spite of its subtle null phenotype, Drosophila retain abundant potential
to reveal how huntingtin does its job in neurons, particularly with respect to
axonal transport. The facility associated with observation and measurement of in
vivo trafficking of fluorescently labeled axonal cargos via fluorescent microscopy
is a powerful tool for functional screening of therapeutic compounds emerging as
candidates from cell-based assays, such as Geldanamycin (Herbst, M. and
Wanker, E.E. 2007). The behavioral phenotype observed in Huntingtin mutants
and fly HD models is a valuable marker for identifying relevant genetic factors in
large-scale screens of expanding RNAi and transposon insertion libraries (Dietzl
et al., 2007; Thibault et al., 2004).
APPENDIX A: Genetic schemes
huntingtin gamma ray allele generation
w
(
wPBac*
PBac'* C
'
X w; Tm3
Tm6
1 w; PBac' (
Tm6 -
w;
X
Sb
Tm3-GFP
(white eyes)
X w;
PBac"
w; Tm3-GFP
w; Pbac'
Pbac*
Pbac'
ITm3-GFP
-*
FLP-FRT excision of huntingtin
Chr. I!or III
isov
Y
Y
x
Bal
hs-FLP I
Y
hs-FLP
Dom
hs-FLP
Bal
i sow
Bat
Bat
isow
Heat shock progeny larvae
to activate FLP expression
hs-FL P
isow
sow Dom Dom
7
Y
isow or hs-FL P 3
Y
Bat
Bal
Bal
Initial PCR done in next
generation on males with
putative deficiency
viability, PCR
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