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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
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CA 02543058 2006-04-20
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TRANSGENIC FLIES EXPRESSING MUTANT A[342
BACKGROUND
Alzheimer's disease (AD) is the most common neurodegenerative disorder in
humans.
The disease is characterized by a progressive impairment in cognition and
memory. The
hallmark of AD at the neuropathological level is the extracellular
accumulation of the
amyloid-(3 peptide (A(3) in "senile" plaques, and the intracellular deposition
of neurofibrillary
tangles made of the microtubule-associated protein Tau. In neuronal tissue of
AD patients,
Tau is hyperphosphorylated and adopts pathological conformations evident with
conformation-dependent antibodies. The amyloid -~i peptide is a cleavage
product of the
amyloid precursor protein (APP). In normal individuals, most of A[3 is in a 40-
amino acid
form, but there are also minor amounts of A[3 that are 42 amino acids in
length (A(342). In
patients with AD, there is an overabundance of A(342 that is thought to be the
main toxic A[3
form.
A number of pathogenic' mutations have been found within APP which are
associated
with hereditary forms of AD, several of which are located within the A(3
sequences. These
mutations result in a phenotype different from AD, with massive amyloid
accumulation in
cerebral blood vessel walls. Two mutations, namely the Dutch (G1u22G1n) and
the Flemish
(Ala2lGly) mutations, have been reported (Levy, et al., Science 248, 1124-1126
(1990)),
(van Broeckhoven et al. (1990)), (Hendriks,°et al., Nature Genet 1, 218-
221 (1992)). Patients
having these mutations suffer from cerebral hemorrhage arid vascular symptoms.
The
vascular symptoms are caused by aggregation of A(3 in blood vessel walls
(amyloid
angiopathy). A third pathogenic infra-A~3; mutation was recently discovered in
an Italian
family (G1u22Lys), with clinical findings similar to the Dutch patients
(Tagliavini, et al., Alz
Report 2, S28 (1999)). Yet another pathogenic AD mutation within APP, named
the "Arctic
mutation" (Glu22Gly), is also located within the A(3 peptide domain of the APP
gene.
Garners of this mutation develop progressive dementia with clinical features
typical of AD
without symptoms of cerebrovascular disease. AD is distinctly characterized by
accelerated
formation of protofibrils comprising mutated A(3 peptides (A~i40ARC and/or
A~342ARC)
compared to protofibril formation of wild type A(3 peptides. Finally, carriers
of the "Iowa"
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
mutation, carrying a Asp23Asn mutation within A(3, exhibit severe cerebral
amyloid
angiopathy, widespread neurofibrillary tangles, and unusually extensive
distribution of A(340
in plaques. (Grabowski et al., Ann. Neurol. 49: 691-693 (2001))
A number of transgenic mouse models have been generated that express wild-type
or
mutant human APP. The mutant form of APP is differentially cleaved to result
in increased
amounts of A(342 deposited within A(3 plaques. These transgenic mice present
with
neurological symptoms of Alzheimer's disease, such as impaired memory and
motor function
(Janus C. et al., Curr. Neurol. Neurosci. Rep 1 (5): 451-457 (2001)). A
transgenic mouse that
expresses both mutant human APP and mutant human Tau has also been generated
(Dada, et.
al., Science, (5534) 293:1487-1491 (2001)). This double transgenic mouse is a
rodent model
for AD that shows enhanced neurofibrillary degeneration indicating that either
APP or A[3
influences the formation of neurofibrillary tangles.
Mouse models have proven very useful for testing potential AD therapeutics.
However,
the use of mice for testing therapeutics is both expensive and time consuming.
Thus, it would
be beneficial to find alternative models which are less expensive and that can
be efficiently
used to screen for therapeutic agents for Alzheimer's disease. For example,
non-mammalian
animal models, such as e'aenor~habditis elegarzs or Drosophila fnelanogaste~.
The use of Drosophila as a model organism has proven to be an important tool
in the
elucidation of human neurodegenerative pathways (reviewed in Fortini, M. and
Bonini, N.
Trends Genet. 16: 161-167 (2000)), as the Drosophila genome contains many
relevant human
orthologs that are extremely well conserved in function (Rubin, G. M., et al.,
Science 287:
2204-2215 (2000)). For example, Drosophila melanogaster carries a gene that is
homologous
to human APP which is involved in nervous system function. The gene, APP-like
(Apply, is
~ approximately 40% identical to the neurogenic isoform (Rosen et al., Proc.
Natl. Acad. Sci.
U.S.A. 86:2478-2482 (1988)) and, like human APP695, is exclusively expressed
in the
nervous system. Flies deficient for the Appl gene show behavioral defects
which can be
rescued by the human APP gene, suggesting that the two genes have similar
functions in the
two organisms (Luo et al., Neuron 9:595-605 (1992)).
In addition, Drosophila models of polyglutamine repeat diseases (Jackson, G.
R., et al
(1998). Neuron 21: 633-642; Kazemi-Esfarani, P. and Benzer, S. (2000). Science
287: 1837-
1840; Fernandez-Funez et al. (2000) Nature 408 (6808):101-6), Parkinson's
disease (Feany,
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CA 02543058 2006-04-20
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M. B. and Bender, W. W. (2000). Nature 404: 394-398) and others have been
established
which closely mimic the disease state in humans at the cellular and
physiological levels, and
have been successfully employed in identifying other genes that may be
involved in these
diseases. Thus, the power of Drosophila as a model system is demonstrated in
the ability to
represent the disease state and to perform large scale genetic screens to
identify critical
components of disease. This invention generally relates to a method to
identify compounds
and genes acting on the APP pathway in transgenic Drosophila melanogaster that
ectopically
express genes related to AD. Expression of these transgenes can induce visible
phenotypes
and it is contemplated herein that genetic screens disclosed herein may be
used to identify
genes involved in the APP pathway by the identification of mutations that
modify the induced
visible phenotypes. The genes affected by these mutations will be called
herein "genetic
modifiers". It is contemplated herein that human homologs of such genetic
modifiers would
be useful targets in the development of therapeutics to treat conditions
associated with, but
not limited to, Alzheimer Disease.
SUMMARY OF THE INVENTION
The present invention discloses transgenic flies that express the human A(342
peptide of
APP containing a pathogenic mutation selected from the group consisting of the
'Iowa
mutation' (D23N) within the A(342 (A(342IoWa ) peptide of SEQ ID NO: 1, the
'Dutch
mutation' (E22Q) within the A(342 (A(342D"t~n ) peptide of SEQ ID NO: 2, the
'Flemish
mutation' (A21G) within the A(342 (Aj342Fiemtsh ) peptide of SEQ lD NO: 3, and
the 'Italian
mutation' (E22I~ within the A(342 (A(342Itauan ) peptide of SEQ ID NO: 4.
The present invention discloses transgenic flies that express the human A(342
peptide of
APP containing a pathogenic 'Arctic mutation' (E22G) within the A(342
(A(342A,.~~;~ ) peptide
of SEQ ID NO: 5 and the human Tau protein.
The present invention provides transgenic flies whose somatic and germ cells
comprise a
transgene encoding the human A[342IoWa containing the Iowa mutation, the human
A[342D"toh
containing the Dutch mutation, the human A(342Flem;sh containing the Flemish
mutation, and
the human A(342Ita1ian containing the Italian mutation and wherein expression
of the transgene
results in the fly having a predisposition to, or resulting in, progressive
neural degeneration.
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CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
The present invention provides transgenic flies whose somatic and germ cells
comprise
transgenes encoding the Tau and human A(342A,.~t;~ containing the Arctic
mutation and
wherein expression of the transgene results in the fly having a predisposition
to, or resulting
in, progressive neural degeneration.
In one embodiment, the transgenic fly is transgenic Drosophila.
In a preferred embodiment of the invention, the transgenic fly comprising the
Iowa,
Dutch, Flemish, or Italian A(342 mutation comprises a second transgene,
encoding the Tau
protein. The double transgenic fly of this embodiment as well as the double
transgenic fly
comprising the Arctic A(342 mutation and Tau, displays a synergistic altered
phenotype as
compared to the altered phenotype displayed by transgenic flies expressing
mutant human
A(342 protein alone.
In a more preferred embodiment of this invention, the Tau and human A(342IoWa,
A~342Dutch, A[~42Flemisn, A(I42rtaiian, or A[342A,.~t;~ mutant transgenes are
operatively linked to an
expression control sequence and expression of the transgenes results in an
observable
phenotype. In one embodiment, the transgene is temporally regulated by the
expression
control sequence. In another embodiment, the transgene is spatially regulated
by the
expression control sequence. In a specific embodiment of the invention, the
expression
control sequence is a heat shock promoter. In a preferred mode of the
embodiment, the heat
shock promoter is derived from the hsp70 or hsp83 genes. In other specific
embodiments, the
Tau and human A[342IoWa, A(~42Dutcn~ A(~42Flemisna A(i42Itatian~ or
A(342A,.~ti~ transgenes are
operatively linked to a GAL4. Upstream Activating Sequence ("UAS").
Optionally, the
transgenic D~osophila comprising Tau and human A(342IoWa, A(~42Dut~n,
A(~42~e,,,;sh,
A(342Itauan, or A(342P,L~t;~ mutant transgenes further comprise a GAL4 gene.
In a preferred
embodiment, the GAL4 gene is linked to a tissue specific expression control
sequence. In a
preferred mode of the embodiment, the tissue specific expression control
sequence is derived
from the seve~less, eyeless, gmrlglass or any of the rhodopsin genes. In
another preferred
mode of the embodiment, the tissue specific expression control sequence is
derived from the
dpp, vestigial, or apteYOUS genes. In another preferred mode of the
embodiment, the tissue
specific expression control sequence is derived from neural-specific genes
like elav, hirvarZa
or D42 genes. In yet other embodiments, the expression control sequence is
derived from
ubiquitously expressed genes like tubulin, actin, or ubiquitin. In yet other
embodiments, the
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CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
expression control sequence comprises a tetracycline-controlled
transcriptional activator
(tTA) responsive regulatory element. Optionally, the transgenic Drosophila
comprising the
Tau and mutant human A(342IoWa, A(~42Dutcn~ A[~42F1em;sh, A(342I~1,~,, or
A(342~~;~ transgenes
further comprise a tTA gene.
The DNA sequence encoding the mutant human A(342IoWa, A(~42Dutcn~ A(~42F1e~sn~
A[342Ita1ian~ or A(342~.~~;~ may be fused to a signal peptide, e.g., via an
amino acid linker. The
signal peptide may be a wingless (wg) signal peptide, such as the peptide
represented by SEQ
m NO: 11, or an Argos (aos) signal peptide, such as the sequence of SEQ m NO:
12. The
transgenic fly may exhibit an altered phenotype, such as a rough eye
phenotype, a concave
wing phenotype, a locomotor dysfunction (e.g., reduced climbing ability,
reduced wallcing
ability, reduced flying ability, decreased speed, abnormal traj ectories, and
abnormal
turnings), abnormal grooming, other abnormal behaviors, or reduced life span.
In another aspect, the invention relates to a method for identifying an agent
active in
neurodegenerative disease. The method comprises the steps of (a) providing a
transgenic fly
whose genome comprises DNA sequences that encode the mutant human A(342IoWa,
A(342Dut~h, A~42Flemisha or A(342I~lian alone, or in combination with the Tau
protein, or that
encode the A[342A,.~tt~ in combination with Tau protein; (b) providing a
candidate agent to the
transgenic fly; and (c) observing the phenotype of the transgenic fly of step
(b) relative to the
control fly that has not been administered an agent. An observable difference
in the
phenotype of the transgenic fly that has been administered an agent compared
to the control
fly that has not been administered an agent is indicative of an agent active
in
neurodegenerative disease. In yet another aspect, the invention relates to a
method for
identifying an agent active in neurodegenerative disease. The method comprises
the steps of
(a) providing a transgenic fly and a control wild-type fly; (b) providing a
candidate agent to
the transgenic fly and to the control fly; and (c) observing a difference in
phenotype between
the transgenic fly and the control fly, wherein a difference in phenotype is
indicative of an
agent active in neurodegenerative disease.
In a further aspect, the invention relates to a method to identify genetic
modifiers of the
APP pathway, comprising: providing a transgenic fly whose genome comprises a-
DNA
sequence encoding a polypeptide comprising the A~342IoWa, A(342D"t~h,
A~42Fle~sh~ or
A(342Ita1i~n (SEQ. m NOS: 6, 7, ~, 9, respectively) which is optionally fused
to a signal
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
sequence, alone or together with DNA sequence encoding the Tau, or a DNA
sequence
encoding a polypeptide comprising the A~i42~.~n~ (SEQ ID NO: 10) which is
optionally fused
to a signal sequence, together with DNA sequence encoding the Tau, where the
DNA
sequence is operably linked to a tissue- specific expression control sequence;
and wherein
expression of said DNA sequences) results in an altered phenotype; crossing
the transgenic
fly with a fly containing a mutation in a known or predicted gene; and,
screening progeny for
flies that display modified expression of the transgenic phenotype as compared
to controls.
Experimental techniques for performing the steps involved in the screen
described above are
described, for example, in Cohen et al., (US20020174446A1), or Benzer et al.,
(W0200112238A1), herein incorporated by reference.
DETAILED DESCRIPTION
The present invention discloses transgenic flies that express human A(342IoWa,
containing
a D23N mutation, human A(342Dutch~ containing a E22Q, human A(342Flemisha
containing a
A21 G mutation, or human A(342Itat,a", containing a E22K mutation either alone
or in
combination with the Tau protein, or human A(342A,.~t;~, containing a E22G
mutation in
combination with the Tau protein. The transgenic flies exhibit progressive
neurodegeneration
which can lead to a variety of altered phenotypes including locomotor
phenotypes, behavioral
phenotypes (e.g., appetite, mating behavior, and/or life span), and
morphological phenotypes
(e.g., shape, size, or location of a cell, organ, or appendage; or size,
shape, or growth rate of
the fly).
As used herein, the term "transgenic fly" refers to a fly whose somatic and
germ cells
comprise a transgene operatively linked to a promoter, wherein the transgene
encodes the
human A(342Iowa~ A(~42D"c~n, A(342F~emish, or A[342Ita~ian~ or A(342~.~~;~ in
combination with Tau,
and wherein the expression of said transgenes in the nervous system results in
said
Drosophila having a predisposition to, or resulting in, progressive neural
degeneration. The
term "double transgenic fly" refers to a transgenic fly whose somatic and germ
cells comprise
at least two transgenes, wherein the transgenes encode the Tau and human
A(342toWa,
A~i42D"t~h, Aa42Flemish, A(~42Ita1ian~ or A~i42,~.~t;~. It will be readily
apparent to one of skill in
the art that a "transgenic fly" comprising the human A(342A,.~t;~ mutation
also expresses Tau,
and will thus necessarily be a "double transgenic fly". Although the
exemplified double
transgenic fly is produced by crossing two single transgenic flies, the double
transgenic fly of
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the present invention can be produced using any method known in the art for
introducing
foreign DNA into an animal. The terms "transgenic fly" and "double transgenic
fly" include
all developmental stages of the fly, i.e., embryonic, larval, pupal, and adult
stages. The
development of Drosophila is temperature dependent. The Drosophila egg is
about half a
millimeter long. It takes about one day after fertilization for the embryo to
develop and hatch
into a worm-like larva. The larva eats and grows continuously, molting one
day, two days,
and four days after hatching (first, second and third instaxs). After two days
as a third instar
larva, it molts one more time to form an immobile pupa. Over the next four
days, the body is
completely remodeled to give the adult winged form, which then hatches from
the pupal case
and is fertile after another day (timing of development is for 25°C; at
1 ~°, development takes
twice as long).
As used herein, "fly" refers to an insect with wings, such as Drosophila. As
used herein,
the term "Drosophila" refers to any member of the Drosophilidae family, which
include
without limitation, Drosophila funebris, Drosophila multispina, D~osophila
subfunebris,
guttifera species group, Drosophila guttifera, Drosophila albomicans,
Drosophila annulipes,
Drosophila curviceps, Drosophila formosana, Drosophila hypocausta, Drosophila
inZmigrans, Drosophila keplauana, Drosophila kohkoa, Drosophila nasuta,
Drosophila
neohypocausta, Drosophila niveifrons, Drosophila pallidiftons, Drosophila
pulaua,
Drosophila quadrilineata, Drosophila siamana, Drosophila sulfurigaster
albostrigata,
Drosophila sulfurigaster bilimbata, Drosophila sulfurigaster neonasuta,
Drosophila Taxora
F, Drosophila Taxon I, Drosophila ustulata, Drosophila melanica, Drosophila
paramelanica,
DrosoplZila tsigarza, Drosophila daruma, Drosophila polyehaeta, quinaria
species group,
Drosophila falleni, Drosophila nigromaculata, Drosophila palustris, Drosophila
phalerata,
Drosophila subpalustris, Drosophila eohydei, Drosophila hydei, Drosophila
lacertosa,
Drosophila robusta, Drosophila sordidula, Drosophila repletoides, Drosophila
kanekoi,
Drosophila virilis, Drosophila maculinatata, Drosophila ponera, Drosoplaila
ananassae,
Drosophila atripex, Drosophila bipeetinata, Drosophila ercepeae, Drosophila
malerkotliana
malerkotliana, Drosophila malerkotliana pallens, Drosophila parabipectinata,
Drosophila
pseudoananassae pseudoananassae, Drosophila pseudoananassae nigrens,
Drosophila
varians, Drosoplaila elegans, Drosophila gunungcola, Drosophila eugracilis,
Drosophila
fieusphila, Drosophila erecta, Drosophila mauritiana, Drosophila melanogaster,
Drosophila
orena, Drosophila sechellia, Drosophila simulans, Drosophila teissieri,
Drosophila yakuba,
Drosophila auraria, Drosophila baimaii, Dr-osophila barbarae, Drosophila
biauraria,
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Drosophila birchii, Drosophila bocki, Drosophila bocqueti, Drosophila burlai,
Drosophila
constricts (sensu Chen & Okada), Drosophila jambulina, Drosophila khaoyana,
Drosophila
kikkawai, Drosophila lacteicornis, Drosophila leontia, Drosophila lini,
Drosophila mayri,
Drosophila parvula, Drosophila pectinifera, Drosoplaila punjabiensis,
Drosophila quadraria,
Drosophila rufa, Drosophila seguyi, Drosophila serrate, Drosophila subauraria,
Drosophila
tarsi, Drosophila trapezifrons, Drosophila triauraria, Dr osophila truncate,
Drosophila
vulcaraa, Drosophila watanabei, Drosophila fuyamai, Drosophila biarmipes,
Drosophila
mimetica, Drosophila pulchrella, Drosophila suzukii, Drosophila uraipectinata,
Drosophila
lutescens, Drosophila paralutea, Drosophila prostipennis, Drosophila
takahashii,
Drosophila trilutea, Drosophila bifasciata, Drosophila imaii, Df-osophila
pseudoobscura,
Drosophila saltans, Drosophila sturtevanti, Drosophila nebulosa, Drosophila
paulistorum,
and Drosophila willistoni. In one embodiment, the fly is Drosophila
melanogaster.
As used herein, "A(342IoWa, A~342Dut~n, A(342F~emish~ A~42ltatian, ~d
A(~42A,.~t;~" is used to
refer to a mutant form of the 42-amino acid polypeptide that is produced in
nature through the
proteolytic cleavage of human amyloid precursor protein (APP) by beta and
gamma
secretases. A/342IoWa, A(I42Dutch, A(I42FIe",ish~ A(~42lta~ian, or
A(342~,,.~~;~ differs from wildtype
A(342 in that it contains a Asp23Asn mutation (SEQ ID NO: 1), a G1u22G1n
mutation (SEQ
ll~ NO: 2), a A1a21 Gly mutation (SEQ ID NO: 3), a G1u22Lys mutation (SEQ ID
NO: 4), or
a G1u22G1y mutation (SEQ ID NO: 5), respectively. A(342 is a major component
of
extracellular amyloid plaque depositions found in neuronal tissue of
Alzheimer's disease
patients. In the present invention, A(342IoWa, A(~42Dutcha A(~42~emish~
A(~42Italian, or A(342~".~h~
includes a peptide encoded by a recombinant DNA wherein a nucleotide sequence
encoding
Aa42Io~,a, Aa42Dutch~ A~42F1emish~ Aa42ltaliane Or A(342A,.~~;~ is operatively
linked t0 an
expression control sequence such that the A[342IoWa, A(~42Dutch, A(~42Ftemish~
A(~42Ita1ian~ or
A(342~.~t;~ peptide is produced in the absence of cleavage of APP by beta and
gamma
secretase. It is noted that, because of the degeneracy of the genetic code,
different nucleotide
sequences can encode the same polypeptide sequence.
As used herein, the term "amyloid plaque depositions" refers to insoluble
protein
aggregates that are formed extracellularly by the accumulation of amyloid
peptides, such as
A(342.
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
As used herein, the term "signal peptide" refers to a short amino acid
sequence, typically
less than 20 amino acids in length, which directs proteins to or through the
endoplasmic
reticulum secretory pathway of Dr~osophila. For example, "signal peptides"
include, but are
not limited to, the Drosophila signal peptides of Dint protein synonymous to
"wingless (wg)
signal peptide" (SEQ ID NO: 11) and the "Argos (aos) signal peptide" (SEQ ID
NO: 12), the
D~osophila Appl (SEQ ID NO: 13), presenilin (SEQ ID NO: 14), or windbeutel
(SEQ m
NO: 1 S). Any conventional signal sequence that directs proteins through the
endoplasmic
reticulum secretory pathway, including variants of the above mentioned signal
peptides, can
be used in the present invention.
As used herein, an "amino acid linker" refers to a short amino acid sequence
from about 2
to 10 amino acids in length that is flanked by two individual peptides.
As used herein, the term "tau protein" refers to the microtubule-associated
protein Tau
that is involved in microtubule assembly and stabilization. In neuronal
tissues of Alzheimer's
disease patients, Tau is found in intracellular depositions of neurofibrillary
tangles. The
human gene that encodes the human Tau protein contains 11 exons, and is
described by
Andreadis, A. et al., Biochemistry, 31 (43):10626-10633 (1992), herein
incorporated by
reference. In adult human brain, six tau isoforms are produced from a single
gene by
alternative mRNA splicing. They differ from each other by the presence or
absence of 29- or
58- amino-acid inserts located in the amino-terminal half and 31-amino acid
repeat located in
the carboxyl-terminal half. Inclusion of the latter, which is encoded by exon
10 of the tau
gene, gives rise to the three tau isoforms which each have 4 repeats.
As used herein, the term "Tau protein" includes various Tau isoforms produced
by
alternative mRNA splicing as well as mutant forms of a sequence encoding human
Tau
proteins as described in SEQ ID NO': 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO: 20,
and SEQ 117 21. In one embodiment, the Tau protein used to generate the double
transgenic
fly is represented by SEQ ID NO: 16 (amino acid sequence) and SEQ ID NO: 17
(nucleotide
sequence). In the normal cerebral cortex, there is a slight preponderance of 3
repeat over 4
repeat tau isoforms. These repeats and some adjoining sequences constitute the
microtubule-
binding domain of tau (Goedert, et al., 1998 Neuron 21, 955-958). In neuronal
tissues of
Alzheimer's disease patients, Tau is hyperphosphorylated and adopts abnormal
and/or
pathological conformations detectable using conformational-dependent
antibodies, such as
MCI and ALZ50 (Jicha G.A., et al., Journal of Neuroscience Research 48:128-132
(1997)).
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Thus, "Tau protein", as used herein, includes Tau protein recognized by these
conformation
specific-antibodies.
The invention further contemplates, as equivalents of these Tau sequences,
mutant
sequences that retain the biological effect of Tau of forming neurofibrillary
tangles.
Therefore, "Tau protein", as used herein, also includes Tau proteins
containing mutations and
variants. These mutations include but are not limited to: Exon 10 +12
"Kumamoto pedigree"
(Yasuda et al., (2000) Ann Neurol. 47: 422-9); I260V (Grower et al., Exp
Neurol. 2003 Nov ;
184(1):131-40); G272V (Hutton et al., 1998 Nature 393:702-5; Heutink et al.,
(1997) Ann
Neurol. 41(2):150-9; Spillantini et al., (1996) ActaNeuropathol (Berl). 1996
Ju1;92(1):42-8);
N279K (Clark et al., (1998). Proc Natl Acad Sci USA 95: 13103-13107; D'Souza
et al.,
(1999) Proc Natl Acad Sci USA. 96: 5598-5603; Reed et al., (1997) Ann Neurol.
1997
42:564-72; Hasegawa et al., (1999) FEBS Letters 443: 93-96; Hong et al.,
(1998) Science
282: 1914-1917); de1K280 (Rizzu et al., (1999) Am J Hum Genet 64: 414-421;
D'Souza et
al., (1999) Proc Natl Acad Sci USA. 96: 5598-5603) L284L (D'Souza et al.,
(1999) Proc Natl
Acad Sci USA. 96: 5598-5603); P301L (Hutton et al., 1998 Nature 393:702-5;
Heutink et al.,
(1997) Ann Neurol. 41(2):150-9; Spillantini et al., (1996) Acta Neuropathol
(Berl). 1996
Ju1;92(1):42-8; Hasegawa et al., (1998) FEBS Lett. 1998 437(3):207-101;
Nacharaju et al.,
(1999) FEBS Letters 447: 195-199); P301S Bugiani (1999) J Neuropathol Exp
Neurol.
58:667-77; Goedert et al., (1999) FEBS Letters 450: 306-311); S305N (Iijima et
al., (1999)
Neuroreport 10: 497-501; Hasegawa et al., (1998) FEBS Lett. 1998 437(3):207-
101; D'Souza
et al., (1999) Proc Natl Acad Sci USA. 96: 5598-5603); S305S (Stanford et al.,
Brain, 123,
880-893, 2000) S305S (Wszolek et al., Brain. 2001 124:1666-70); V337M (Poorkaj
et al.,
(1998) Ann Neurol. 1998 43:815-25; Spillantini et al., (1998) American Journal
of Pathology
153: 1359-1363; Sumi et al., (1992) Neurology. 42:120-7; Hasegawa et al.,
(1998) FEBS
Lett. 1998 437(3):207-10); G389R Murrell et al., J Neuropathol Exp Neurol.
1999
Dec;58(12):1207-26; Pickering-Brown, et al., Ann Neurol. 2000 48(6):859-67);
R406W
(Hutton et al., 1998 Nature 393:702-5; Reed et al., (1997) Ann Neurol. 1997
42:564-72;
Hasegawa et al., (1998) FEBS Lett. 1998 437(3):207-101); 3'ExlO+3, GtoA
(Spillantini et
al., (1998) American Journal of Pathology 153: 1359-1363; Spillantini et al.,
(1997) Proc
Natl Acad Sci U S A. 199794(8):4113-8); 3'ExlO+16 (Baker et al., (1997) Annals
of
Neurology 42: 794-798; Goedert et al., (1999b) Nature Medicine 5: 454-457;
Hutton et al.,
(1998) Nature 393: 702-705); 3'ExlO+14 (Hutton et al., (1998) Nature 393: 702-
705; Lynch
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WO 2005/041650 PCT/US2004/034838
et al., (1994) Neurology 44:1878-1884); 3'ExlO+13 (Hutton et al., (1998)
Nature 393: 702-
705).
Many human Tau gene sequences exist. In adult human brain, six tau isoforms
are
produced from a single gene by alternative mRNA splicing (Goedert et al.,
Neuron. 1989
3:519-26). It is noted that, because of the degeneracy of the genetic code,
different nucleotide
sequences can encode the same polypeptide sequence. The invention further
contemplates the
use of Tau genes containing sequence polyrnorphisms (See, for example, Table
1).
Table 1. Polymorphisms identified within the human Tau gene.
Underlined polymorphisms are inherited as a part of extended
haplotype 2. In case of exons skipped in the brain mRNA (exon 4a,
6, 8) locations of polymorphic sites are counted from the first
nucleotide of the exon.
Exon/IntronPolymorphisms
El 5' UTR-13 a--> g
Il nt-93 t --> c
I2 nt+18 c --> t
I3 nt+9 a --> a
I3 nt-103 t --> a (very rare on H1)
I3 nt-94a -->t (very rare on H1)
E4a n+232 C --> T (CCG/CTG; P/L)
E4a n+480 G --> A (GAC/AAC; R/N)
E4a n+482 C --> T (GAC/GAT; N/N)
E4a n+493 T --> C (GTA/GCA; V/A)
E4a n316 A --> G (CAA/CGA, Q/Q)
I4a nt-72 t --> c .
E6 n+139 C --> T (CAC/TAC H/Y) (very
common)
E6 n+157 T --> C (ACT/ACC S/P)
I6 nt+67 a --> a
I6 nt+105 t --> c
E7 P176P (G --> A)
E8 n+5 T --> C (ACT/ACC, T/T)
I8 nt-26 g~> a
E9 A227A GCA/GCG)
E9 N255N AAT/AAC)
E9 P270P (CCG/CCA)
I9 nt-47 c --> a (very rare on H1)
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I9 02- 3gbp
I1l nt+34 g
--> a
Ill nt+90 g
--> a
Ill nt+296 c
--> t
I13 nt+34 t
--> c
The invention also contemplates the use of Tau proteins or genes from other
animals,
including but not limited to mice (Lee et al., (1988) Science 239, 285-8),
rats (Goedert et al.,
(1992) Proc. Natl. Acid. Sci. U.S.A. 89 (5); 1983-1987), Bos tauYUS (Himmler
et al., (1989)
Mol. Cell. Biol. 9 (4), 1381-1388), Drosoplaila melahogaste~ (Heidary &
Fortini, (2001)
Mech. Dev. 108 (1-2), 171-178) andXenopus laevis (Olesen et al., (2002) Gene
283 (1-2),
299-309). The Tau genes from other animals may additionally contain mutations
equivalent
to those previously described. Equivalent positions can be identified by
sequence alignment,
and equivalent mutations can be introduced by means of site-directed
mutagenesis or other
means known in the art.
As used herein, the term "neurofibrillary tangles" refers to insoluble twisted
fibers that
form intracellularly and that are composed mainly of Tau protein.
As used herein, the term "operatively linked" refers to a juxtaposition
wherein the
components described are in a relationship permitting them to function in
their intended
manner. An expression control sequence "operatively linked" to a coding
sequence is ligated
in such a way that expression of the coding sequence is achieved under
conditions compatible
with the activity of the control sequences.
As used herein, the term "expression control sequence" refers to promoters,
enhancer
elements, and other nucleic acid sequences that contribute to the regulated
expression of a
given nucleic acid sequence. The term "promoter" refers to DNA sequences
recognized by
RNA polymerise during initiation of transcription and can include enhancer
elements. As
used herein, the term "enhancer element" refers to a cis-acting nucleic acid
element, which
controls transcription initiation from homologous as well as heterologous
promoters
independent of distance and orientation. Preferably, an "enhancer element"
also controls the
tissue and temporal specification of transcription initiation. In particular
embodiments,
enhancer elements include, but are not limited to, the UAS control element.
"UAS" as used
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WO 2005/041650 PCT/US2004/034838
herein, refers to an Upstream Activating Sequence recognized and bound by the
Gal4
transcriptional activator. The term "UAS control element", as used herein,
refers to a UAS
element that is activated by Gal4 transcriptional regulator protein. A "tissue
specific"
expression control sequence, as used herein, refers to expression control
sequences that drive
expression in one tissue or a subset of tissues, while being essentially
inactive in at least one
other tissue. "Essentially inactive" means that the expression of a sequence
operatively
linked to a tissue specific expression control sequence is less than 5% of the
level of
expression of that sequence in that tissue where the expression control
sequence is most
active. Preferably, the level of expression in the tissue is less than 1 % of
the maximal
activity, or there is no detectable expression of the sequence in the tissue.
"Tissue specific
expression control sequences" include those that are specific for organs such
as the eye, wing,
notum, brain, as well as tissues of the central and peripheral nervous
systems. Examples of
tissue specific control sequences include, but are not limited to, the
sevehless
promoter/enhancer (Bowtell et al., Genes Dev. 2(6):620-34 (1988)); the eyeless
promoter/enhancer (Bowtell et al., Proc. Natl. Acad. Sci. U.S.A. 88(15):6853-7
(1991));
gmrlglass responsive promoters/enhancers (Quiring et al., Science 265:785-9
(1994)), and
promoterslenhancers derived from any of the rhodopsin genes, that are useful
for expression
in the eye; enhancers/promoters derived from the dpp or vestigial genes useful
for expression
in the wing (Staehling-Hampton et al., Cell Growth Differ. 5(6):585-93
(1994)); Kim et al.,
Nature 382:133-8 (1996)); promoters/enhancers derived from elav (Yao and
White, J.
Neurochem. 63(1):41-51 (1994)), Appl (Martin-Morris and White, Development
110(1): 185-
95 (1990)), and niYVaha (Sun et al., Proc. Nat'1 Acad. Sci. U.S.A. 96: 10438-
43 (1999)) genes
useful for expression in the central nervous system; and promoters/enhancers
derived from
neural specific D42 genes, all of which references are incorporated by
reference herein.
Other examples of expression control sequences include, but are not limited to
the heat shock
promoters/enhancers from the hsp70 and hsp83 genes, useful for temperature
induced
expression; and promoters/enhancers derived from ubiquitously expressed genes,
such as
tubulin, actin, or Ubiquitin.
As used herein, the term "phenotype" refers to an observable and/or measurable
physical,
behavioral, or biochemical characteristic of a fly. The term "altered
phenotype" as used
herein, refers to a phenotype that has changed relative to the phenotype of a
wild-type fly.
Examples of altered phenotypes include a behavioral phenotype, such as
appetite, mating
behavior, and/or life span, that has changed by a measurable amount, e.g. by
at least 10%,
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20%, 30%, 40%, or more preferably 50%, relative to the phenotype of a control
fly; or a
morphological phenotype that has changed in an observable way, e.g. different
growth rate of
the fly; or different shape, size, color, or location of an organ or
appendage; or different
distribution, and/or characteristic of a tissue, as compared to the shape,
size, color, location of
organs or appendages, or distribution or characteristic of a tissue observed
in a control fly.
As used herein, "a synergistic altered phenotype" or "synergistic phenotype,"
refers to a
phenotype wherein a measurable and/or observable physical, behavioral, or
biochemical
characteristic of a fly is more than the sum of its components.
A "change in phenotype" or "change in altered phenotype," as used herein,
means a
measurable and/or observable change in a phenotype relative to the phenotype
of a control
fly.
As used herein, the "rough eye" phenotype is characterized by irregular
ommatidial
packing, occasional ommatidial fusions, and missing bristles that can be
caused by
degeneration of neuronal cells. The eye becomes rough in texture relative to
its appearance
in wild type flies, and can be easily observed by microscope.
As used herein, the "concave wing" phenotype is characterized by abnormal
folding of
the fly wing such that wings are bent upwards along their long margins.
As used herein, "locomotor dysfunction" refers to a phenotype where flies have
a deficit
in motor activity or movement (e.g., at least a 10% difference in a measurable
parameter) as
compared to control flies. Motor activities include flying, climbing,
crawling, and turning.
In addition, movement traits where a deficit can be measured include, but are
not limited to:
i) average total distance traveled over a defined period of time; ii) average
distance traveled
in one direction over a defined period of time; iii) average speed (average
total distance
moved per time unit); iv) distance moved in one direction per time unit; v)
acceleration (the
rate of change of velocity with respect to time; vi) turning; vii) stumbling;
viii) spatial
position of a fly to a particular defined area or point; ix) path shape of the
moving fly; and x)
undulations during larval movement; xi) rearing or raising of larval head; and
xii) larval tail
flick. Examples of movement traits characterized by spatial position include,
without
limitation: (1) average time spent within a zone of interest (e.g., time spent
in bottom, center,
or top of a container; number of visits to a defined zone within container);
and (2) average
distance between a fly and a point of interest (e.g., the center of a zone).
Examples of path
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WO 2005/041650 PCT/US2004/034838
shape traits include the following: (1) angular velocity (average speed of
change in direction
of movement); (2) turning (angle between the movement vectors of two
consecutive sample
intervals); (3) frequency of turning (average amount of turning per unit of
time); and (4)
stumbling or meander (change in direction of movement relative to the
distance). Turning
parameters can include smooth movements in turning (as defined by small
degrees rotated)
and/or rough movements in turning (as defined by large degrees rotated).
As used herein, a "control fly" refers to a larval or adult fly of the same
genotype of the
transgenic fly as to which it is compared, except that the control fly either
i) does not
comprise one or both of the transgenes present in the transgenic fly, or ii)
has not been
administered a candidate agent.
As used herein, the term "candidate agent" refers to a biological or chemical
compound
that when administered to a transgenic fly has the potential to modify the
phenotype of the
fly, e.g. partial or complete reversion of the altered phenotype towards the
phenotype of a
wild type fly. "Agents" as used herein can include any recombinant, modified
or natural
nucleic acid molecule, library of recombinant, modified or natural nucleic
acid molecules,
synthetic, modified or natural peptide, library of synthetic, modified or
natural peptides; and
any organic or inorganic compound, including small molecules, or library of
organic or
inorganic compounds, including small molecules.
As used herein, the term "small molecule" refers to compounds having a
molecular mass
of less than 3000 Daltons, preferably less than 2000 or 1500, more preferably
less than 1000,
and most preferably less than 600 Daltons. Preferably but not necessarily, a
small molecule
is a compound other than an oligopeptide.
As used herein, a "therapeutic agent" refers to an agent that ameliorates one
or more of
the symptoms of a neurodegenerative disorder such as Alzheimer's disease in
mammals,
particularly humans. A therapeutic agent can reduce one or more symptoms of
the disorder,
delay onset of one or more symptoms, or prevent or cure the disease.
EXAMPLES
I. Generation of Transgenic Drosophila
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
A transgenic fly that carries a transgene that encodes the mutant A~42IoWa,
A(342Dutch~
A~34~Flemish~ A~4~Itaiian, or A(342A,.~c;~ (plus Tau) as well as a double
transgenic fly carrying
both the Tau protein and the mutant human A(342IoWa, Aa42p"tch, A(342Flemish,
A(l4~itan~~ or
A(342A,.~h~ peptide are disclosed. The transgenic flies provide a model for
neurodegenerative
disorders such as Alzheimer's disease, which is characterized by an
extracellular
accumulation of A(342IoWa peptide and an intracellular deposition of a
hyperphosphorylated
form of microtubule-assaciated protein Tau. The transgenic flies of the
present invention can
be used to screen for therapeutic agents effective in the treatment of
Alzheimer's disease.
A. General
The transgenic flies of the present invention can be generated by any means
known to
those skilled in the art. Methods for production and analysis of transgenic
Drosophila strains
are well established and described in Brand et al., Methods in Cell Biology
44:635-654
(1994); Hay et al., Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997); and in
Robert D.B.
Drosophila: A Practical Approach, Washington D.C. (1986), herein incorporated
by
reference in their entireties.
In general, to generate a transgenic fly, a transgene of interest is stably
incorporated into a
fly genome. Any fly can be used, however a preferred fly of the present
invention is a
member of the Drosophilidae family. An exemplary fly is Drosophila
Melanogaster.
A variety of transformation vectors are useful for the generation of the
transgenic flies of
the present invention, and include, but are not limited to, vectors that
contain transposon
sequences, which mediate random integration of transgene into the genome, as
well as
vectors that use homologous recombination (Bong and Golic, Science 288: 2013-
2018
(2000)). A preferred vector of the present invention is pUAST (Brand and
Perrimon,
Development 118:401-415 (1993)) that contains sequences from the transposable
P-element
which mediate insertion of a transgene of interest into the fly genome.
Another preferred
vector is PdL that is able to yield doxycycline-dependent overexpression
(Nandis, Bhole and
Tower, Genome Biology 4 (R8):1-14, (2003)).
P-element transposon mediated transformation is a commonly used technology for
the
generation of transgenic flies and is described in detail in Spradling, P
element mediated
transformation, In Drosophila: A Practical Approach (ed. D. B. Roberts),
pp#175-197, lRL
16
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Press, Oxford, UK (1986), herein incorporated by reference. Other
transformation vectors
based on transposable elements, include fox example, the hobo element
(Blackman et al.,
Embo J. 8(1):211 -7) (1989)), mariner element (Lidholm et al., Genetics
134(3):859-68
(1993)), the hermes element (O'Brochta et al., Genetics 142(3):907-14 (1996)),
Minos
(Loukeris et al., Proc. Natl. Acad. Sci. USA 92(21):9485-9 (1995)), or the
PiggyBac element
(Handler et al., Proc. Natl. Acad. Sci. USA 95(13):7520-5 (1998)). In general,
the terminal
repeat sequences of the transposon that are required for transposition are
incorporated into a
transformation vector and arranged such that the terminal repeat sequences
flank the
transgene of interest. It is preferred that the transformation vector contains
a marker gene
used to identify transgenic animals. Commonly used, marker genes affect the
eye color of
Drosophila, such as derivatives of the Drosophila white gene (Pirrotta V., &
C. Brockl,
EMBO J. 3(3):563-8 (1984)) or the Drosophila rosy gene (Doyle W. et al., Eur.
J Biochem.
239(3):782-95 (1996)) genes. Any gene that results in a reliable and easily
measured
phenotypic change in transgenic animals can be used as a marker. Examples of
other marker
genes used for transformation include the yellow gene (Wittkopp P. et al.,
Curr Biol.
12(18):1547-56 (2002)) that alters bristle and cuticle pigmentation; the
forked gene
(McLachlan A., Mol Cell Biol. 6(1):1-6 (1986)) that alters bristle morphology;
the Adh+
gene used as a selectable marker for the transformation of Adh- strains
(McNabb S. et al.,
Genetics 143(2):897-911 (1996)); the Ddc+ gene used to transform Ddctsa mutant
strains
(Scholnick S. et al., Cell 34(1):37-45(1983)); the lacZgene ofE. coli; the
heonaycinRgefze
from the E. coli transposon TnS; and the green fluorescent protein (GFP;
Handler and Harrell,
Insect Molecular Biology 8:449-457 (1999)), which can be under the control of
different
promoter/enhancer elements, e.g. eyes, antenna, wing and leg specific
promoter/enhancers, or
the poly-ubiquitin promoter/enhancer elements.
Plasmid constructs for introduction of the desired transgene are coinjected
into
Drosophila embryos having an appropriate genetic background, along with a
helper plasmid
that expresses the specific transposase needed to mobilize the transgene into
the genomic
DNA. Animals arising from the injected embryos (GO adults) are selected, or
screened
manually, for transgenic mosaic animals based on expression of the marker gene
phenotype
and are subsequently crossed to generate fully transgenic animals (G1 and
subsequent
generations) that will stably carry one or more copies of the transgene of
interest.
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Binary systems are commonly used for the generation of transgenic flies, such
as the
UAS/GAL4 system. This system is a well-established which employs the UAS
upstream
regulatory sequence for control of promoters by the yeast GAL4 transcriptional
activator
protein, as described in Brand and Perrimon, Development 118(2):401-15 (1993))
and Rorth
et al, Development 125(6):1049-1057 (1998), herein incorporated by reference
in their
entireties. In this approach, transgenic D~asophila, termed "target" lines,
are generated where
the gene of interest (e.g. A(342Iowa, A(~42Duc~h, A(~42Flemish~ A(~f2Italian~
or A(342~h~ and/or
TAU)) is operatively linked to an appropriate promoter controlled by UAS.
Other transgenic
Df°osophila strains, termed "driver" lines, are generated where the
GAL4 coding region is
operatively linked to promoters/enhancers that direct the expression of the
GAL4 activator
protein in specific tissues, such as the eye, antenna, wing, or nervous
system. The gene of
interest is not expressed in the "target" lines for lack of a transcriptional
activator to "drive"
transcription from the promoter joined to the gene of interest. However, when
the UAS-
target line is crossed with a GAL4 driver line, the gene of interest is
induced. The resultant
progeny display a specific pattern of expression that is characteristic fox
the GAL4 line.
The technical simplicity of this approach makes it possible to sample the
effects of
directed expression of the gene of interest in a wide variety of tissues by
generating one
transgenic target line with the gene of interest, and crossing that target
line with a panel of
pre-existing driver lines. Individual GAL4 driver Drosophila strains with
specific drivers
have been established and axe available for use (Brand and Perrimon,
Development
118(2):401-15 (1993)). Driver strains include, for example apterous-Gal4
(wings, brain,
interneurons), elav-Gal4 (CNS), sevenless-Gal4, eyeless-Gal4, GMR-Gal4 (eyes)
and the
brain specific 7B-Gal4 driver.
B Generation of transgenic flies
The present invention discloses transgenic flies that have incorporated into
their genome a
DNA sequence that encodes a mutant human A(342IoWa, A(342D"t~h, A(342F1em;sh,
A[342ltati~n~ or
A(342A,.~t;~ fused to a signal peptide, as well as double transgenic flies
which comprise a DNA
sequence that encodes the Tau protein as well as a DNA sequence encoding the
mutant '
human A(342xoWa, A(342Duc~h, A(342Flemish~ Aa42itanan, or A~342,~,.~tic used
to a signal peptide.
Generation of transgenic flies containing single transgenes can be performed
using any
standard means known to those skilled in the art. To generate the double
transgenic fly,
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transgenic Drosophila that express either the A(342IoWa, A(342Dut~n,
A(342F~emish~ or A~342I~lian,
or the Tau protein, or A(342,~.~~;o and the Tau protein are independently made
and then crossed
to generate a Drosophila that expresses both proteins.
In a preferred embodiment, transgenic Drosophila are produced using the
UAS/GAL4
control system. Briefly, to generate a transgenic fly that expresses Tau, a
DNA sequence
encoding Tau is cloned into a vector such that the sequence is operatively
linked to the GAL4
responsive element UAS. Vectors containing UAS elements are commercially
available,
such as the pUAST vector (Brand and Perrimon, Development 118:401-415 (1993)),
which
places the UAS sequence element upstream of the transcribed region. The DNA is
cloned
using standard methods (Sambrook et al., Molecular Biology. A laboratory
Approach, Cold
Spring Harbor, N.Y. (1989); Ausubel, et al., Curreht protocols in Moleeular
Biology, Greene
Publishing, Y, (1995)) and is described in more detail under the Molecular
Techniques
section of the present application. After cloning the DNA into appropriate
vector, such as
pUAST, the vector is injected into Drosophila embryos (e.g. yw embryos) by
standard
procedures (Brand et al., Methods in Cell Biology 44:635-654 (1994)); Hay et
al., Proc. Natl.
Acad. Sci. USA 94(10):5195-200 (1997) to generate transgenic Drosophila.
When the binary UAS/GAL4 system is used, the transgenic progeny can be crossed
with
Drosophila driver strains to assess the presence of an altered phenotype. A
preferred
Drosoplaila comprises the eye specific driver strain gmr-GAL4, which enables
identification
and classification of transgenics flies based on the severity of the rough eye
phenotype.
Expression of Tau in Drosophila eye results in the rough eye phenotype
(characterized by an
eye with irregular ommatidial packing, occasional ommatidial fusions, and
missing bristles),
which can be easily observed by microscope. The severity of the rough eye
phenotype
exhibited by a transgenic line can be classified as strong, medium, or weak.
The weak or
mild lines have a rough, disorganized appearance covering the ventral portion
of the eye.
The medium severity lines show greater roughness over the entire eye, while in
strong
severity lines the entire eye seems to have lost/fused many of the ommatidia
and
interommatidial bristles, and the entire eye has a smooth, glossy appearance.
To generate a transgenic fly that expresses the mutant human A(342, a DNA
sequence
encoding human A~342IoWa, A(342Dutcn~ A(~42~~emisn, A(~42Italian, or
A(342p,,.~no is ligated in frame
to a DNA sequence encoding a signal peptide such that the A~i42IoWa,
Aa42Dutcha A(~42F~emish~
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A(342Italian~ or A(342~.on~ peptide can be exported across cell membranes. The
signal sequence
is directly linked to the A~342IoWa, A(342D"t~h, A[342Fiemisna A(~42Itatian,
or A(342A,.otic coding
sequence or indirectly linked by using a DNA linker sequence, for example of
3, 6, 9, 12, or
15 nucleotides. A signal peptide that directs proteins to or through the
endoplasmic reticulum.
secretory pathway of D~osophila is used. Preferred signal peptides of the
present invention
are the Argos (aos) signal peptide (SEQ m NO: 12), the wingless (wg) signal
peptide (SEQ
m NO: 11) the DYOSOphila Appl (SEQ m NO: 13), presenilin (SEQ m NO: 14), and
windbeutel (SEQ DJ NO: 15).
The DNA encoding the mutant A(342IoWa, A(342D"toh, Aa42F1er";Sh, A(342ltaiian~
or A(342,~,.~t;~
peptide is linked to a signal sequence by standard ligation techniques and is
then cloned into a
vector such that the sequence is operatively linked to the GAL4 responsive
element UAS. A
preferred transformation vector for the generation of A(342IoWa, A(342Dutcn~
A(~42~emisn~
A(342Ita1ian, or A(342A,.~t;~ transgenic flies is the pUAST vector (Brand and
Perrimon,
Development 11 x:401-415 (1993)). As described for the generation of Tau
transgenic flies,
the vector is injected into Drosophila embryos (e.g. yw embryos) by standard
procedures
(Brand et al., Meth. in Cell Biol. 44:635-654 (1994)); Hay et al., Proc. Natl.
Acad. Sci. USA
94(10):5195-200 (1997)) and progeny are then selected and crossed based on the
phenotype
of the selected marker gene. When the binary UAS/GAL4 system is used, the
transgenic
progeny can be crossed with Drosophila driver strains to assess the presence
of an altered
phenotype. Preferred Drosophila driver strains are gmY-GAL4 (eye) and elav-
GAL4 (CNS).
Without being bound to one particular theory, it is believed that the ectopic
overexpression of the A(342 and/or Tau sequences described herein leads to
neurodegeneration which can have numerous cellular, physiological, behavioral
and
morphological effects. For example, neurodegeneration in the eye is believed
to give rise to
the rough eye phenotype; neurodegeneration in the wing (i.e., neuromuscular
degeneration) is
believed to give rise to morphological wing abnormalities such as the concave
wing; and
neurodegeneration in the CNS or PNS is believed to give rise to numerous
locomotive and
behavioral phenotypes. Abberant overexpression of the A(342 and/or Tau
sequences .
described herein may be evaluated by screening flies for phenotypic changes
which are
commensurate with the tissue-specific expression of the sequence as dictated
by a particular
expression control sequence. For example, were the gmy; severaless, eyeless,
or rhodopsin-
derived eye-specific promoter/enhancer is used to direct expression in the
eye, a phenotype
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such as the rough-eye phenotype is expected to be observed. Where an
enhancer/promoter
derived from the dpp or vestigial genes is used to direct expression in the
wing, a phenotype
such as the concave wing is expected to be obaerved. Where a promoter/enhancer
derived
from elav, Appl, or fziYVaraa is used to direct expression in the central
nervous system, or a
promoter/enhancer derived from neural specific D42 genes is used,
neurological, locomotor,
andlor behavioral phenotypes can be expected to be obeserved. The converse
approach is
also contemplated. For example, to assess an eye phenotype (e.g., rough eye
phenotype) a
gmr-GAL4 driver strain is used in the cross. Ectopic overexpression of mutant
A(342IoWa,
A(342D"t~h, A(342F1emisn~ A.(342Ita1ian~ or A(342,~.~~;~ in D~osophila eye is
believed to disrupt the
regular trapezoidal arrangement of the photoreceptor cells of the ommatidia
(identical single
units, forming the Drosophila compound eye), the severity of which is believed
to depend on
transgene copy number and expression levels. To evaluate a locomotor phenotype
(e.g.,
climbing assay), an elau (or other neural specific promoter)-Gal4 driver
strain is used in the
cross. Ectopic overexpression of mutant A(342IoWa, A(~42Dutch, A(~42Flemish~
A(~42Italian~ or
A(342~.~~;~ in D~osophila central nervous system (CNS) is believed to result
in locomotor
deficiencies, such as impaired movement, climbing and flying. To evaluate a
wing
phenotype (e.g., concave wing), a dpp- or vestigial-Gal4 driver strain is used
in the cross.
Ectopic overexpression of mutant A[342jOWa, A(342Dutch, A(342Fiemisha
A(~42I,~tian, or A(342~,t.~n~ in
D~osophila wing is believed to result in a concave wing phenotype, evidenced
by abnormal
folding of the fly wing such that wings are bent upwards along their long
margins.
Once the single transgenic flies are produced, the flies can ~be crossed with
each other by
mating. Flies are crossed according to conventional methods. When the binary
UAS/GAL4
system is used, the fly is crossed with an appropriate driver strain and the
altered phenotype
assessed, as described above, transgenic flies are classified by assessing
phenotypic severity.
For example, as disclosed herein, the combination of Tau and mutant A(342IoWa,
~1(~42Dut~h~
A(342F1e~"isn~ A(342ltaaan, or A(342Ar~n~ transgenes is believed to produce a
synergistic effect on
the eye.
Expression of Tau and mutant A(342IoWa, A~42Dutch, A(~42F1emisn, A(~42It~1ian,
or A(342~.~~;~
proteins in transgenic flies is confirmed by standard techniques, such as
Western blot analysis
or by immunostaining of Drosophila tissue cross-sections, both of which are
described
below.
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a. Western Blot analysis
Western blot analysis is performed by standard methods. Briefly, as means of
example,
to detect expression of the A/342 peptide or Tau by western blot analysis,
whole flies, or
D~osophila heads (e.g. 80-90 heads) are collected and placed in an eppendorf
tube on dry ice
containing 100 p.1 of 2% SDS, 30% sucrose, 0.718 M Bistris, 0. 318 M Bicine,
with
"Complete" protease inhibitors (Boehringer Mannheim), then ground using a
mechanical
homogenizer. Samples are heated for 5 min at 95° C, spun down for 5 min
at 12,000 rpm,
and supernatants are transferred into a fresh eppendorf tube. 5% (3-
mercaptoethanol and
0.01% bromphenol blue are added and samples are boiled prior to loading on a
separating
gel. Approximately 200 ng of total protein extract is loaded for each sample,
on a 15%
Tricine/Tris SDS PAGE gel containing 8M Urea. After separating, samples are
then
transferred to PVDF membranes (BIO-RAD, 162-0174) and the membranes are
subsequently
boiled in PBS for 3 min. Anti-Tau antibody (e.g. T14 (Zymed) and AT100 (Pierce-
Endogen)
or anti-~i42 antibody (e.g. 6E10 (Senetek PLC Napa, CA.) are hybridized,
generally at a
concentration of 1:2000, in 5% non-fat milk, 1 ~ PBS containing 0.1% Tween 20,
for 90 min
at room temperature. Samples are washed 3 times 'for 5 min., 15 min. and 15
min. each, in 1 X
PBS-0.1% Tween-20. Labeled secondary antibody, (for example, anti-mouse-HRP
from
Amersham Pharmacia Biotech, NA 931) is prepared, typically at a concentration
of 1:2000,
in 5% non- fat milk, 1 ~ PBS containing 0.1% Tween 20, for 90 min at room
temperature.
Samples are then washed 3 times for 5 min., 15 min. and 15 min. each, in 1 ~
PBS-0.1
Tween- 20. Protein is then detected using the appropriate method. For example,
when anti-
mouse-HRP is used as the conjugated secondary antibody, ECL (ECL Western
Blotting
Detection Reagents, Amersham Pharnlacia Biotech, # RPN 2209) is used for
detection.
b. Cross sections
As a manner of confirming protein expression in transgenic flies,
imrnunostaining of
1)~osophila organ cross sections is performed. Such a method is of particular
use to confirm
the presence of hyperphosphorylated Tau, which is a modified form of the Tau
protein that is
present in non-diseased tissue. Hyperphosphorylated Tau exhibits altered
pathological
conformations as compared to Tau protein and is present in diseased tissue
from patients with
certain neurodegenerative disorders, such as Alzheimer's disease.
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Cross sections of Drosophila organs can be made by any conventional
cryosectioning,
such as the method described in Wolff, Drosophila Protocols, CSHL Press
(2000), herein
incorporated by reference. Cryosections can then be immunostained for
detection of Tau and
A(342 peptides using methods well known in the art. In a preferred embodiment,
the
Vectastain ABC Kit (which comprises biotinylated anti-mouse IgG secondary
antibody, and
avidinlbiotin conjugated to the enzyme Horseradish peroxidase H (Vector
Laboratories) is
used to identify the protein. In other embodiments the secondary antibody is
conjugated to a
fluorophore. Briefly, cryosections are blocked using normal horse serum,
according to the
Vectastain ABC Kit protocol. The primary antibody, recognizing the human A(342
peptide or
Tau, is typically used at a dilution of 1:3000 and incubation with the
secondary antibody is
done in PBS/1%BSA containing 1-2% normal horse serum, also according to the
Vectastain
ABC Kit protocol. The procedure for the ABC Kit is followed; incubations with
the ABC
reagent axe done in PBS/0.1°!° saponin, followed by 410 minute
washes in PBS/0.1%
saponin. Sections are then incubated in 0.5 ml per slide of the Horseradish
Peroxidase H
substrate solution, 400 ug/ml 3,3'-diaminobenzidene (DAB), 0.006% H 202 in
PBS/0.1%
saponin, and the reaction is stopped after 3 min. with 0.02% sodium azide in
PBS. Sections
are rinsed several times in PBS and dehydrated through an ethanol series
before mounting in
DPX (Fluka).
Exemplary antibodies that can be used to immunostain cross sections include
but are not
limited to, the monoclonal antibody 6E10 (Senetek PLC Napa, CA.) that
recognizes A(342
peptide and anti-Tau antibodies ALZSO and MCI (Jicha GA, et al., J. of
Neurosci. Res.
48:128-132 (1997)).
Alternatively, antibodies for use in the present invention that recognize
A(342 and Tau
can be made using standard protocols known in the art (See, for example,
Antibodies: A
Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A
mammal,
such as a mouse, hamster, or rabbit can be immunized with an immunogenic form
of the
protein (e.g., a A(342 or Tau polypeptide or an antigenic fragment Which is
capable of
eliciting an antibody response). Immunogens for raising antibodies are
prepared by mixing
the polypeptides (e.g., isolated recombinant polypeptides or synthetic
peptides) with
adjuvants. Alternatively, A(342 or Tau polypeptides or peptides are made as
fusion proteins
to larger immunogenic proteins. Polypeptides can also be covalently linked to
other larger
immunogenic proteins, such as keyhole limpet hemocyanin. Alternatively,
plasmid or viral
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vectors encoding A(342 or Tau, or a fragment of these proteins, can be used to
express the
polypeptides and generate an immune response in an animal as described in
Costagliola et al.,
J. Clin. Invest. 105:803-811 (2000), which is incorporated herein by
reference. In order to
raise antibodies, immunogens are typically administered intradermally,
subcutaneously, or
intramuscularly to experimental animals such as rabbits, sheep, and mice. In
addition to the
antibodies discussed above, genetically engineered antibody derivatives can be
made, such as
single chain antibodies.
The progress of immunization can be monitored by detection of antibody titers
in
plasma or serum. Standard ELISA, flow cytometry or other immunoassays can also
be used
with the immunogen as antigen to assess the levels of antibodies. Antibody
preparations can
be simply serum from an immunized animal, or if desired, polyclonal antibodies
can be
isolated from the serum by, for example, affinity chromatography using
immobilized
immunogen.
To produce monoclonal antibodies, antibody-producing splenocytes can be
harvested
from an immunized animal and fused by standard somatic cell fusion procedures
with
immortalizing cells such as myeloma cells to yield hybridoma cells. Such
techniques are
well known in the art, and include, for example, the hybridoma technique
(originally
developed by Kohler and Milstein, Nature, 256: 495-497 (1975)), the human B
cell
hybridoma technique (Kozbar et al., Immunology Today, 4: 72 (1983)), and the
EBV-
hybridoma technique to produce human monoclonal antibodies (Cole et al.,
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96(1985)). Hybridoma
cells can be
screened immunochemically for production of antibodies that are specifically
reactive with
A(342 or Tau peptide, or polypeptide, and monoclonal antibodies isolated from
the media of a
culture comprising such hybridoma cells.
II. Molecular Techniques
In the present invention, DNA sequences that encode Tau or human A(342IoWa,
A[~42Dutch,
A(342F,emish~ A(J42ltaiian~ or A(342Ar~tt~ are cloned into transformation
vectors suitable for the
generation of transgenic flies.
A. Generation of DNA sequences encoding Tau or human A(342
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DNA sequences encoding Tau and A(342IoWa, A~342Du~~n, A(342F1emisn~
A(~42ltatian~ or
A(342~~;~ can be obtained from genomic DNA or be generated by synthetic means
using
methods well known in the art (Sambrook et al., Molecular Biology: A
laboratory Approach,
Cold Spring Harbor, N.Y. (1989); Ausubel, et al., Current protocols in
Molecular Biology, f
Greene Publishing, Y, (1995)). Briefly, human genomic DNA can be isolated from
peripheral blood or mucosal scrapings by phenol extraction, or by extraction
with kits such as
the QIAamp Tissue kit (Qiagen, Chatsworth, Cal.), Wizard genomic DNA
purification kit
(Promega, Madison, Wis.), and the ASAP genomic DNA isolation kit (Boehringer
Mannheim, Indianapolis, Ind.). DNA sequences encoding Tau and A(342IoWa,
A~i42Dut~h,
A~4'2Flemish~ A(~42Itatiana or A(342~.~c;~ can then be amplified from genomic
DNA by
polymerase chain reaction (PCR) (Mullis and Faloona Methods Enzymol., 155: 335
(1987)),
herein incorporated by reference) and cloned into a suitable recombinant
cloning vector.
Alternatively, a cDNA that encodes Tau or human A[342zoWa, A(~42Dutcn,
A(~42Ftemish~
A(342ltatian~ or A(342~.~t;~ can be amplified from mRNA using RT-PCR and
cloned into a
suitable recombinant cloning vector. RNA may be prepared by any number of
methods
known in the art; the choice may depend on the source of the sample. Methods
for preparing
RNA axe described in Davis et al., Basic Methods in Molecular Biology,
Elsevier, NY,
Chapter 11 (1986); Ausubel et al., Current Protocols in Molecular Biology,
Chapter 4, John
Wiley and Sons, NY (1987); Kawasaki and Wang, PCR Technology, ed. Erlich,
Stockton
Press NY (1989); Kawasaki, PCR Protocols: A Guide to Methods and Applications,
Innis et
al. eds. Academic Press, San Diego (1990); all of which are incorporated
herein by
reference.
It is preferred, following generation of sequences that encode Tau or
A(342IoWa, A(342D"tch~
Aa42glemish, A(J42uatian~ or A(342~.~tt~ by PCR or RT-PCR, that the sequences
are cloned into
an appropriate sequencing vector in order that the sequence of the cloned
fragment can be
confirmed by nucleic acid sequencing in both directions,
Suitable recombinant cloning vectors for use in the present invention contain
nucleic acid
sequences that enable the vector to replicate in one or more selected host
cells. Typically in
cloning vectors, this sequence is one that enables the vector to replicate
independently of the
host chromosomal DNA and includes origins of replication or autonomously
replicating
sequences. Such sequences are well known for a variety of bacteria, yeast and
viruses. For
CA 02543058 2006-04-20
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example, the origin of replication from the plasmid pBR322 is suitable for
most Gram-
negative bacteria, the 2 micron plasmid origin is suitable for yeast, and
various viral origins
(e.g. SV40, adenovirus) are useful for cloning vectors in mammalian cells.
Generally, the
origin of replication is not needed for mammalian expression vectors unless
these are used in
mammalian cells able to replicate high levels of DNA, such as COS cells.
Advantageously, a cloning or expression vector may contain a selection gene
also
referred to as a selectable marker. This gene encodes a protein necessary for
the survival or
growth of transformed host cells grown in a selective culture medium. Host
cells not
transformed with the vector containing the selection gene will therefore not
survive in the
culture medium. Typical selection genes encode proteins that confer resistance
to antibiotics
and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline,
complement
auxotrophic deficiencies, or supply critical nutrients not available in the
growth media.
Since cloning is most conveniently performed in E. coli, an E. coli-selectable
marker, for
example, the (3-lactamase gene that confers resistance to the antibiotic
ampicillin, is of use.
These can be obtained from E. coli plasmids, such as pBR322 or a pUC plasmid
such as
pUC 18 or pUC 19.
Sequences that encode Tau or human A~342Iowa, A~42Dutch~ A(~42Flemish~
A[~~2italian~ or
A[34-2~.~t;~ can also be directly cloned into a transformation vector suitable
for generation of
transgenic Drosophila such as vectors that allow for the insertion of
sequences in between
transposable elements, or insertion downstream of an UAS element, such as
pUAST. Vectors
suitable for the generation of transgenic flies preferably contain marker
genes such that the
transgenic fly can be identified such as, the white gene, the rosy gene, the
yellow gene, the
forked gene, and others mentioned previously. Suitable vectors can also
contain tissue
specific control sequences as described earlier, such as, the set~enless
promoter/enhancer, the
eyeless promoter/enhancer, glass-responsive promoters (gmr)/enhancers useful
for expression
in the eye; and enhancers/promoters derived from the dpp or vestigial genes
useful for
expression in the wing.
Sequences that encode Tau or human A(342IoWa, Aa42Dut~r,, A(342FIemisha
A(~42itaii~n~ or
A(342,~.~t;~ are ligated into a recombinant vector in such a way that the
expression control
sequences are operatively linked to the coding sequence.
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Herein, DNA sequences that encode Tau or human A[342IoWa, A[342Dutch~
A~42F~emish~
A(342Italian, or A(342,d,,.~n~ can be generated through the use of Polymerase
chain reaction
(PCR), or RT-PCR which uses RNA-directed DNA polymerase (e.g., reverse
transcriptase) to
synthesize cDNAs which is then used for PCR.
III. Phenotypes and methods of detecting altered phenotypes
A double transgenic fly according to the invention can exhibit an altered eye
phenotype,
of progressive neurodegeneration in the eye that leads to measurable
morphological changes
in the eye (Fernandez-Funez et al., Nature 408:101-106 (2000); Steffan et. al,
Nature
413:739-743 (2001)). The Drosophila eye is composed of a regular trapezoidal
arrangement
of seven visible rhabdomeres produced by the photoreceptor neurons of each
Drosophila
ommatidium. A phenotypic eye mutant according to the invention leads to a
progressive loss
of rhabdomeres and subsequently a rough-textured eye. A rough textured eye
phenotype is
easily observed by microscope or video camera. In a screening assay for
compounds which
alter this phenotype, one may observe slowing of the photoreceptor
degeneration and
improvement of the rough-eye phenotype (Steffan et. al, Nature 413:739-743
(2001)).
A transgenic or double transgenic fly according to the invention can exhibit
an altered
wing phenotype, believed to be rooted in neuromuscular degeneration in the
wing, leading to
measurable morphological changes in the wing structure. A concave wing
phenotype may be
easily observed by microscope, video camera, or other suitable imaging means.
Neuronal degeneration in the central nervous system will give rise to
behavioral deficits,
including but not limited to locomotor deficits, that can be assayed and
quantitated in both
larvae and adult Drosophila. For example, failure of Drosophila adult animals
to climb in a
standard climbing assay (see, e.g. Ganetzky and Flannagan, J. Exp. Gerontology
13:189-196
(1978); LeBourg and Lints, J. Gerontology 28:59-64 (1992)) is quantifiable,
and indicative of
the degree to which the animals have a motor deficit and neurodegeneration.
Neurodegenerative phenotypes include, but are not limited to, progressive loss
of
neuromuscular control, e.g. of the wings; progressive degeneration of general
coordination;
progressive degeneration of locomotion, and progressive loss of appetite.
Other aspects of
Drosoplaila behavior that can be assayed include but are not limited to
circadian behavioral
rhythms, feeding behaviors, inhabituation to external stimuli, and odorant
conditioning. All
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of these phenotypes are measured by one skilled in the art by standard visual
observation of
the fly.
Another neural degeneration phenotype, is a reduced life span, for example,
the
Drosophila life span can be reduced by 10-80%, e.g., approximately, 30%, 40%,
50%, 60%,
or 70%. Any observable and/or measurable physical or biochemical
characteristic of a fly is
a phenotype that can be assessed according to the present invention.
Transgenic flies can be
produced by identifying flies that exhibit an altered phenotype as compared to
control (e.g.,
wild-type flies, or flies in which the transgene is not expressed).
Therapeutic agents can be
identified by screening for agents, that upon administration, result in a
change in an altered
phenotype of the transgenic fly as compared to a transgenic fly that has not
been administered
a candidate agent.
A change in an altered phenotype includes either complete or partial reversion
of the
phenotype observed. Complete reversion is defined as the absence of the
altered phenotype,
or as 100°~o reversion of the phenotype to that phenotype observed in
control flies. Partial
reversion of an altered phenotype can be 5%, 10%, 20%, preferably 30%, more
preferably
50%, and most preferably greater than 50% reversion to that phenotype observed
in control
flies. Example measurable parameters include, but are not limited to, size and
shape of
organs, such as the eye; distribution of tissues and organs; behavioral
phenotypes (such as,
appetite and mating); and locomotor ability, such as can be observed in a
climbing assays.
For example, in a climbing assay, locomotor ability can be assessed by placing
flies in a vial,
knocking them to the bottom of the vial, then counting the number of flies
that climb past a
given mark on the vial during a defined period of time. 100% locomotor
activity of control
flies is represented by the number of flies that climb past the given mark,
while flies with an
altered locomotor activity can have 80%, 70%, 60%, SO%, preferably less than
50%, or more
preferably less than 30% of the activity observed in a control fly population.
Locomotor
phenotypes also can be assessed as described in provisional application
60/396,339, Methods
for Identifying Biologically Active Agents, herein incorporated by reference.
Briefly,
locomotor dysfunction phenotypes which may be measured according to the
invention
include deficits in motor activity or movement (e.g., at least a 10%
difference in a measurable
parameter) as compared to control flies. Motor activities include flying,
climbing, crawling,
and turning. In addition, movement traits where a deficit can be measured
include, but are
not limited to: i) average total distance traveled over a defined period of
time; ii) average
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distance traveled in one direction over a defined period of time; iii) average
speed (average
total distance moved per time unit); iv) distance moved in one direction per
time unit; v)
acceleration (the rate of change of velocity with respect to time; vi)
turning; vii) stumbling;
viii) spatial position of a fly to a particular defined area or point; ix)
path shape of the moving
fly; and x) undulations during larval movement; xi) rearing or raising of
larval head; and xii)
larval tail flick. Examples of movement traits characterized by spatial
position include,
without limitation: (1) average time spent within a zone of interest (e.g.,
time spent in bottom,
center, or top of a container; number of visits to a defined zone within
container); and (2)
average distance between a fly and a point of interest (e.g., the center of a
zone). Examples
of path shape traits include the following: (1) angular velocity (average
speed of change in
direction of movement); (2) turning (angle between the movement vectors of two
consecutive
sample intervals); (3) frequency of turning (average amount of turning per
unit of time); and
(4) stumbling or meander (change in direction of movement relative to the
distance). Turning
parameters can include smooth movements in turning (as defined by small
degrees rotated)
andlor rough movements in turning (as defined by large degrees rotated).
Locomoter defects
in a fly may be measured using methods known in the art, or by taking
measurements
including, but not limited to:
a) total distance (average total distance traveled over a defined period of
time);
b) ~ only distance (average distance traveled in X direction over a defined
period of time;
c) Y only distance (average distance traveled in Y direction over a defined
period of
time);
d) average speed (average total distance moved per time unit);
e) average X-only speed (distance moved in X direction per time unit);
f) average Y-only speed (distance moved in Y direction per time unit);
g) acceleration (the rate of change of velocity with respect to time);
h) turning;
i) stumbling;
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j) spatial position of one animal to a particular defined area or point
(examples of spatial
position traits include (1) average time spent within a zone of interest
(e.g., time spent in
bottom, center, or top of a container; number of visits to a defined zone
within container); (2)
average distance between an animal and a point of interest (e.g., the center
of a zone); (3)
average length of the vector connecting two sample points (e.g., the line
distance between
two animals or between an animal and a defined point or object; e.g. climbing
data); (4)
average time the length of the vector connecting the two sample points is less
than, greater
than, or equal to a user defined parameter; and the like);
m) path shape of the moving animal, i.e., a geometrical shape of the path
traveled by the
animal (examples of path shape traits include the following: (1) angular
velocity (average
speed of change in direction of movement); (2) turning (angle between the
movement vectors
of two consecutive sample intervals); (3) frequency of turning (average amount
of turning per
unit of time); (4) stumbling or meandering (change in direction of movement
relative to the
distance); and the like. This is different from stumbling as defined above.
Turning
parameters may include smooth movements in turning (as defined by small
degrees rotated)
and/or rough movements in turning (as defined by large degrees rotated).
Mernor~r Assay
In Dlrosophila, the best characterized assay for associative learning and
memory is an
odor-avoidance behavioral task (T. Tully, et al. J. Comp. Physiol. A157, 263-
277 (1985),
incorporated herein by reference). This classical (Pavlovian) conditioning
involves exposing
the flies to two odors (the conditioned stimuli, or CS), one at a time, in
succession. During
one of these odor exposures (the CS+), the flies are simultaneously subjected
to electric
shock (the unconditioned stimulus, or IJS), whereas exposure to the other odor
(the CS-)
lacks this negative reinforcement. Following training, the flies are then
placed at a'choice
point, where the odors come from opposite directions, and expected to decide
which odor to
avoid. By convention, leaxning is defined as the fly's performance when
testing occurs
immediately after training. A single training trial produces strong learning:
a typical response
is that >90% of the flies avoid the CS+. Performance of wild-type flies from
this single-cycle
training decays over a roughly 24-hour period until flies once again
distribute evenly between
the two odors. Flies can also form long-lasting associative olfactory
memories, but normally
this requires repetitive training regimens.
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IV. Utility of transgenic flies
A. Disease Model
The transgenic flies of the invention provide a model for neurodegeneration as
is found in
human neurological diseases such as Alzheimer's and tauopathies, such as
Amyotrophic
lateral sclerosis/ parkinsonism-dementia complex of Guam Argyrophilic grain
dementia,
Corticobasal degeneration, Dementia pugilistica, Diffuse neurofibrillary
tangles with
calcification, Frontotemporal dementia with Parkinsonism linked to chromosome
17 (FTDP-
17), Pick's disease, Progressive subcortical gliosis, Progressive supranuclear
palsy (PSP),
Tangle only dementia, Creutzfeldt-Jakob disease, Down syndrome, Gerstmann-
Straussler-
Scheinker disease, Hallervorden-Spatz disease, Myotonic dystrophy, Age-related
memory
impairment, Alzheimer's disease , Amyotrophic lateral sclerosis, Amyotrophic
lateral/parkinsonism-dementia complex of Guam, Auto-immune conditions (eg
Guillain-
Barre syndrome, Lupus), Biswanger's disease, Brain and spinal tumors
(including
neurofibromatosis), Cerebral amyloid angiopathies (Journal of Alzheimer's
Disease vol. 3,
65-73 (2001)), Cerebral palsy, Chronic fatigue syndrome, Creutzfeldt-Jacob
disease
(including variant form), Corticobasal degeneration, Conditions due to
developmental
dysfunction of the CNS parenchyma, Conditions due to developmental dysfunction
of the
cerebrovasculature, Dementia - mufti infarct, Dementia - subcortical, Dementia
with Lewy
bodies, Dementia of human immunodeficiency virus (HIV), Dementia lacking
distinct
histology, Dendatorubopallidolusian atrophy, Diseases of the eye, ear and
vestibular systems
involving neurodegeneration (including macular degeneration and glaucoma),
Down's
syndrome, Dyskinesias (Paroxysmal) Dystonias, Essential tremor, Fahr's
syndrome,
Friedrich's ataxia, Fronto-temporal dementia and Parkinsonism linked to
chromosome 17
(FTDP-17), Frontotemporal lobar degeneration, Frontal lobe dementia, Hepatic
encephalopathy, I3ereditary spastic paraplegia, Huntington's disease,
Hydrocephalus,
Pseudotumor Cerebri and other conditions involving CSF dysfunction, Gaucher's
disease,
Spinal Muscular Atrophy (Hirayama Disease, Werdnig-Hoffinan Disease, Kugelberg-
Welander Disease), Korsakoff's syndrome, Machado-Joseph disease, Mild
cognitive
impairment, Monomelic Amyotrophy, Motor neuron diseases, Multiple system
atrophy,
Multiple sclerosis and other demyelinating conditions (eg leukodystrophies),
Myalgic
encephalomyelitis, Myotonic dystrophy, Myoclonus Neurodegeneration induced by
chemicals, drugs and toxins, Neurological manifestations of Aids including
Aids dementia,
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Neurological conditions (any) arising from polyglutamine expansions,
Neurological /
cognitive manifestations and consequences of bacterial and/or virus
infections, including but
not restricted to enteroviruses, Niemann-Pick disease, Non-Guamanian motor
neuron disease
with neurofibrillary tangles, Non-ketotic hyperglycinemia, Olivo-ponto
cerebellar atrophy,
Opthalinic and otic conditions involving neurodegeneration, including macular
degeneration
and glaucoma, Parkinson's disease, Pick's disease, Polio myelitis including
non-paralytic
polio, Primary lateral sclerosis, Prion diseases including Creutzfeldt-Jakob
disease, kuru, fatal
familial insomnia, and Gerstmann-Straussler-Scheinker disease, prion protein
cerebral
amyloid angiopathy, Postencephalitic Parkinsonism, Post-polio syndrome, Prion
protein
cerebral amyloid angiopathy, Progressive muscular atrophy, Progressive bulbar
palsy,
Progressive supranuclear palsy, Restless leg syndrome, Rett syndrome, Sandhoff
disease,
Spasticity, Spino-bulbar muscular atrophy (Kennedy's disease), Spinocerebellar
ataxias,
Sporadic fronto-temporal dementias, Striatonigral degeneration, Subacute
sclerosing
panencephalitis, Sulphite oxidase deficiency, Sydenham's chorea, Tangle only
dementia,
Tay-Sach's disease, Tourette's syndrome, Transmissable spongiform
encephalopathies,
Vascular dementia, and Wilson disease.
B. Methods for identifying Therapeutic agents
The present invention further provides a method for identifying a therapeutic
agent for
neurodegenerative disease using the transgenic flies disclosed herein. As used
herein, a
"therapeutic agent" refers to an agent that ameliorates the symptoms of
neurodegenerative
disease as determined by a physician. For example, a therapeutic agent can
reduce one or
more symptoms of neurodegenerative disease, delay onset of one or more
symptoms, or
prevent, or cure.
To screen for a therapeutic agent effective against a neurodegenerative
disorder such as
disease, a candidate agent is administered to a transgenic fly. The transgenic
fly is then
assayed for a change in the phenotype as compared to the phenotype displayed
by a control
transgenic fly that has not been administered a candidate agent. An observed
change in
phenotype is indicative of an agent that is useful for the treatment of
disease.
A candidate agent can be administered by a variety of means. For example, an
agent can
be administered by applying the candidate agent to the D~osophilcz cultuxe
media, for
example by mixing the agent in DYOSOphila food, such as a yeast paste that can
be added to
32
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
I~f~osophila cultures. Alternatively, the candidate agent can be prepared in a
1 % sucrose
solution, and the solution fed to D~osophila for a specified time, such as 10
hours, 12 hours,
24 hours, 48 hours, or 72 hours. In one embodiment, the candidate agent is
microinjected
into Drosophila hemolymph, as described in WO 00/37938, published June 29,
2000. Other
modes of administration include aerosol delivery, for example, by vaporization
of the
candidate agent.
The candidate agent can be administered at any stage of Drosophila development
including fertilized eggs, embryonic, larval and adult stages. In a preferred
embodiment, the
candidate agent is administered to an adult fly. More preferably, the
candidate agent is
administered during a larval stage, for example by adding the agent to the
D~osophila culture
at the third larval instar stage, which is the main larval stage in which eye
development takes
place.
The agent can be administered in a single dose or multiple doses. Appropriate
concentrations can be determined by one skilled in the art, and will depend
upon the
biological and chemical properties of the agent, as well as the method of
administration. For
example, concentrations of candidate agents can range from 0.0001 ~.M to 20 mM
when
delivered orally or through inj ection, 0.1 ~,M to 20 mM, 1 ~,M-10 mM, or 10
~,M to 5 mM.
For efficiency of screening the candidate agents, in addition to screening
with individual
candidate agents, the candidate agents can be administered as a mixture or
population of
agents, for example a library of agents. As used herein, a "library" of agents
is characterized
by a mixture more than 20, 100, 1 O3, 104, 105, 106, 108, 1012, or 1015
individual agents. A
"population of agents" can be a library or a smaller population such as, a
mixture less than 3,
5, 10, or 20 agents. A population of agents can be administered to the
transgenic flies and the
flies can be screened for complete or partial reversion of a phenotype
exhibited by the
transgenic flies. When a population of agents results in a change of the
transgenic fly
phenotype, individual agents of the population can then be assayed
independently to identify
the particular agent of interest.
In a preferred embodiment, a high throughput screen of candidate agents is
performed in
which a large number of agents, at least 50 agents, 100 agents or more are
tested individually
in parallel on a plurality of fly populations. A fly population contains at
least 2, 10, 20, 50,
100, or more adult flies or larvae. In one embodiment, locomotor phenotypes,
behavioral
33
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
phenotypes (e.g. appetite, mating behavior, and/or life span), or
morphological phenotypes
(e.g., shape size, or location of a cell, or organ, or appendage; or size
shape, or growth rate of
the fly) are observed by creating a digitized movie of the flies in the
population and the
movie is analyzed for fly phenotype.
B. Candidate Agents
Agents that are useful in the screening assays of the present inventions
include biological
or chemical compounds that when administered to a transgenic fly have the
potential to
modify an altered phenotype, e.g. partial or complete reversion of the
phenotype. Agents
include any recombinant, modified or natural nucleic acid molecule; library of
recombinant,
modified or natural nucleic acid molecules; synthetic, modified or natural
peptides; library of
synthetic, modified or natural peptides; organic or inorganic compounds; or
library of organic
or inorganic compounds, including small molecules. Agents can also be linked
to a common
or unique tag, which can facilitate recovery of the therapeutic agent.
Example agent sources include, but are not limited to, random peptide
libraries as well as
combinatorial chemistry-derived molecular library made of D-and/or L-
configuration amino
acids; phosphopeptides (including, but not limited to, members of random or
partially
degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al.,
Cell 72:767- 778
(1993)); antibodies (including, but not limited to, polyclonal, monoclonal,
humanized, anti-
idiotypic, chimeric or single chain antibodies, and FAb, F(ab')2 and FAb
expression library
fragments, and epitope-binding fragments thereof); and small organic or
inorganic molecules.
Many libraries are known in the art that can be used, e.g. chemically
synthesized libraries,
recombinant libraries (e.g., produced by phage), and in vitro translation-
based libraries.
Examples of chemically synthesized libraries are described in Fodor et al.,
Science 251:767-
773 (1991); Houghten et al., Nature 354:84-86 (1991); Lam et al., Nature
354:82-84 (1991);
Medyuski, Bio/Technology 12:709-710 (1994); Gallop et al., 3. Medicinal
Chemistry
37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 5 90:
10922-10926
(1993); Erb et al., Proc. Natl. Acad. Sci. USA 91:11422-11426 (1994); Houghten
et al.,
Biotechniques 13:412 (1992); Jayawickreme et al., Proc. Natl. Acad. Sci. USA
91:1614-1618
(1994); Salmon et al., Proc. Natl. Acad. Sci. USA 90:11708-11712 (1993); PCT
Publication
No. WO 93/20242; and Brenner and Lerner, Proc. Natl. Acad. Sci. USA 89:5381-
5383
34
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
(1992). By way of examples of nonpeptide libraries, a benzodiazopine library
(see e.g.,
Benin et al., Proc. Natl. Acad. Sci. USA 91:4708-4712 (1994)) can be adapted
for use.
Peptoid libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371
(1992)) can also
be used. Another example of a library that can be used, in which the amide
functionalities in
peptides have been permethylated to generate a chemically transformed
combinatorial
library, is described by Ostreshet al. Proc. Natl. Acad. Sci. USA 91:11138-
11142 (1994).
Examples of phage display libraries wherein peptide libraries can be produced
are described
in Scott & Smith, Science 249:386-390 (1990); Devlin et al., Science, 249:404-
406 (1990);
Christian et al., J. Mol. Biol. 227:711-718 (1992); Lenska, J. hnmunol. Meth.
152:149-157
(1992); Kay et al., Gene 128:59-65 (1993); and PCT Publication No. WO 94/18318
dated
August 18, 1994.
Agents that can be tested and identified by methods described herein can
include, but are
not limited to, compounds obtained from any commercial source, including
Aldrich
(Milwaukee, W1 53233), Sigma Chemical (St. Louis, MO), Fluka Chemie AG (Buchs,
Switzerland) Fluka Chemical Corp. (Ronkonkoma, NY;), Eastman Chemical Company,
Fine
Chemicals (Kingsport, TN), Boehringer Mannheim GmbH (Mannheim, 25 Germany),
Takasago (Rockleigh, NJ), SST Corporation (Clifton, NJ), Ferro (Zachary, LA
70791),
Riedel-deHaen Aktiengesellschaft (Seelze, Germany), PPG Industries Inc., Fine
Chemicals
(Pittsburgh, PA 15272). Further any kind of natural products may be screened
using the
methods described herein, including microbial, fungal, plant or animal
extracts.
Furthermore, diversity libraries of test agents, including small molecule test
compounds,
may be utilized. For example, libraries may be commercially obtained from
Specs and
BioSpecs B.V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego,
CA),
Contract Service Company (Dolgoprudoy, Moscow Region, Russia), Comgenex USA
Inc.
(Princeton, NJ), Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom),
and
Asinex (Moscow, Russia).
Still further, combinatorial library methods known in the art, can be
utilized, including,
but not limited to: biological libraries; spatially addressable parallel solid
phase or solution
phase libraries; synthetic library methods requiring deconvolution; the "one-
bead one-
compound" library method; and synthetic library methods using affinity
chromatography
selection. The biological library approach is limited to peptide libraries,
while the other
CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
approaches are applicable to peptide, non-peptide oligomer or small molecule
libraries of
compounds (Lam, Anticancer Drug Des.l2: 145 (1997)). Combinatorial libraries
of test
compounds, including small molecule test compounds, can be utilized, and may,
for example,
be generated as disclosed in Eichler & Houghten, Mol. Med. Today 1:174-180
(1995); Dolle,
Mol. Divers. 2:223-236 (1997); and Lam, Anticancer Drug Des. 12:145-167
(1997).
Examples of methods for the synthesis of molecular libraries can be found in
the art, for
example in: DeWitt et al., Proc. Natl. Acad. Sci. USA 90:6909 (1993); Erb et
al., Proc. Natl.
Acad. Sci. USA 91:11422 (1994); Zuckermann et al., 3. Med. Chem. 37:2678
(1994); Cho et
al., Science 261:1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl.
33:2059 (1994);
Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061 (1994); and Gallop et al.,
15 J. Med.
Chem. 37:1233 (1994).
A library of agents can also be a library of nucleic acid molecules; DNA, RNA,
or
analogs thereof. For example, a cDNA library can be constructed from mRNA
collected
from a cell, tissue, organ or organism of interest, or genomic DNA can be
treated to produce
appropriately sized fragments using restriction endonucleases or methods that
randomly
fragment genomic DNA. A library containing RNA molecules can be constructed,
for
example, by collecting RNA from cells or by synthesizing the RNA molecules
chemically.
Diverse libraries of nucleic acid molecules can be made using solid phase
synthesis, which
facilitates the production of randomized regions in the molecules. If desired,
the
randomization can be biased to produce a library of nucleic acid molecules
containing
particular percentages of one or more nucleotides at a position in the
molecule (U.S. Pat. No.
5,270,163).
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WO 2005/041650 PCT/US2004/034838
EXAMPLES
Example 1. Generation of transgLenic flies
A transgenic D~osophila anelaizogastef° strain containing a transgene
encoding Tau and a
transgenic Drosophila melafiogaste~ strain containing a transgene encoding
human A[342IoWa,
A(342D"~n, A~42Flemish, A(142ltaiian~ or A(342~.~~;~ peptide are generated as
described herein.
The two transgenic fly strains axe then crossed to obtain a double transgenic
Drosophila
melahogaster strain containing both Tau and human A(342IoWa, A(142Dutcn~
A(~42F~emisn~
A~42Italian~ or A(342,~,.~ri~ genes.
Trans~ene constructs
The UAS/GAL4 system are used to generate both the A(342IoWa, A(342Dut~n,
A~42F~em;sn~ A(~42ltaiian~ or A(342~,z.~t,~ and Tau transgenic flies. A cDNA
encoding the longest
human brain Tau isoform is cloned using standard ligation techniques (Sambrook
et al.,
Molecular Biology: A labor~atoay Approach, Cold Spring Harbor, N.Y'. 1989)
into vector
pUAST (Brand and Perrimon, Development 118:401-415 (1993)) as an EcoRI
fragment in
order to generate transformation vector, pUAS:aNa.RTauwt. The Tau isoform,
which is
represented by SEQ m NO: 17 (nucleic acid sequence), and SEQ m NO: 16 (amino
acid
sequence) contains Tau exons 2 and 3 as well as four microtubule-binding
repeats.
Two pUAST transformation vectors caxrying DNA sequences encoding the
A(342IoWa,
A(342Dut~h, A(342F1emish~ A(142I~tia", or A(342~.~~;~ peptide (SEQ ID NO: 6,
7, 8, 9, or 10,
respectively) are generated. One vector encodes A(342IoWa, A(~42Dutch~
Aaq'2Flemish~ A(34'2Italiana
or A(342,4,.~t;~ peptide fused to the (pUAS:wg-A~342) and another vector
encodes A(342IoWa
peptide fused to Argos (aos) signal peptide (pUAS:aos-A(342). To generate
pUAS:wg-A(342,
a DNA sequence encoding A(342IoWa, A~42D"c~n, A(342F~emisn, A(~42rta~;a", or
A(342~,,.~t,~ peptide
is first fused, in frame, to a synthetic oligonucleotide encoding the wingless
(wg) signal
peptide using a 4 amino acid linker (SFAM). The resulting DNA sequence is then
cloned as
an EcoRI fragment into vector pUAST (Brand and Pernmon, Development 118:401-
415
(1993).
To generate pUAS:aos-A(342, the Argos (aos) signal peptide (SEQ ID NO: 12) is
PCR
amplified from DNA encoding Argos and ligated in frame, to DNA encoding
A(342IoWa,
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CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
A(342nutch~ A~4'2Flemisha A(342ltatian~ or A(342~.~~;~ in the absence of a
linker sequence. The
DNA encoding Argos (aos) signal peptide fused in frame to A~342Iowa,
A(342Dutch~ A(342Flemish~
A~i42ita~,a", or A(342t~,.~n~ is cloned into pUAST (Brand and Perrimon,
Development 118:401-
415 (1993)) as an EcoRI fragment.
Trans~enic Strains
To generate transgenic Drosophila lines expressing either Tau or A(342IoWa,
A(342Dutch~
Aa4'2Flemish~ A~f2Italiam or A~i42P,,.~~;~ the pUAST constructs described
above, either
pUAS:aos-A(342, or pUAS:zrraRTauwt are injected into aylwll$ Drosophila
Melanogaster
embryos as described in (Rubin and Spradling, Science 218:348-353, 1982).
In the case of pUAS:z~4RTauwt, 6 transgenic lines axe generated and classified
by visual
inspection, as described herein, as strong, medium, and weak based on the
severity of the eye
phenotype observed after crossing with a gear-GAL4 driver strain.
In the ease of pUAS:aos- A(342Io,~a, A(342Dut~h~ A(~42Fle~sha A(~42ltai,~", or
A(342,~,.~n~,
transgenic lines are generated and are classified as strong, medium, and weak
based on the
severity of the eye phenotype observed after crossing with a gmr-GAL4 driver
strain.
Transgenic Drosophila strains of moderate eye phenotype that carry the gmr-
GAL4
driver and pUAS:aos-A(342IoWa, A[342Du~~h, A(342Flemish~ ~1.~i42ita~ian, or
A[342A,.~~;~ or
pUAS:zrr4RTauwt are then crossed to generate a double transgenic Drosophila
line that
express both Tau and human A(342Iowa, A(342D~t~n, A(342F1en,;sh, A(342ltanan,
or A(342A,.~n~
peptide. Crossing the single transgenic flies of moderate eye phenotype should
result in a
synergistic eye phenotype classified as strong.
W the case of transformation construct pUAS:wg-A(342, transgenic lines are
generated by
injecting the construct into aylwll$ Dro.sophila Melahogaster embryos as
described in (Rubin
and Spradling, Science 218:348-353, 1982) and screened for the insertion of
transgene into
genomic DNA by monitoring eye color. The pUAST vector carries the white gene
marker.
Transgenic Drosophila carrying wg-A(342IoW~, A~i42Dutch~ A(I42Flemish~
A(~42Itauan~ or A(342.~.~ri~
transgene are then crossed with elav-Gal4 driver strains for expression of the
transgene in the
central nervous system. If the crosses do not result in a measurable
phenotype, the transgene
is mobilized for expansion of copy number by crossing Transgenic Drosophila
carrying wg-
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CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
A(342Iowa, A(~42Dutcn~ A~42Flemish, A(~42itaiian, or A(342~.ot;°
transgene with Df~osophila that
carry a source of P-element. Progeny from this cross are selected based on a
change in eye
color. Flies carrying higher copy numbers of wg- A(342Iowa, A(~42D"ton,
A[342~e",isn~ A(~42Itauan,
or A(342p,,.~t;~ transgene are then crossed with elav-Gal4 driver strains and
locomotor ability of
the crossed flies is tested in climbing assays. Transgenic lines may exhibit a
locomotor
phenotype and the flies are classified as strong, medium, weak and very weak
(28 lines) as
compared among themselves and to elav-Gal4 driver control flies.
A double transgenic DYOSOphala carrying wg- A(342Iowa, A(~42Dutch,
A(342Ftemisn, A(342itauan~
or A(342A,.ot;~ and Tauwt transgenes is then generated by crossing a Tauwt
transgenic
DrosoplZila carrying an elav-Gal4 driver, with an >-vvg- A(342Iowa,
A(~42Dutcn~ A~42Flemisn~
A(342Ita1ian~ or A(342A,.ot;° transgenic D~osophila carrying an elav-
Gal4 driver. Locomotor
ability is assessed and classified as strong, medium, weak and very weak as
compared to
elav-Gal4 driver control flies.
Climbing performance as a function of age is determined for populations of
flies of various
genotypes at 27°C. Climbing assays axe performed in duplicate (two
groups of 30 individuals
of the same age.
Drosophila brain is then cyrosectioned, and horizontal cross sections of elav-
GAL4;
Tauwt/wg- A(342Iowa, A(~42D"t~h, A(342FIemish~ A(~42Ita1ian~ or A(342~,.~t;~
flies are immunostained
with anti-Tau conformation dependent antibodies ALZ50 and MCI. Positive
staining of
neurons may be observed with both MCI antibody (data not shown) and ALZ50
antibody.
The result is expected to show that Tau protein, which is expressed in the
brain of A(342/Tau
double transgenic Drosophila, exhibits protein conformations associated with
Alzheimer's
disease.
Thioflavin-S staining is also performed on cells and neurites of the
transgenic flies,
described herein, to assess the presence of amyloid. Amyloids, when stained
with
Thioflavin-S, fluoresce an apple green color under a fluorescent microscope.
The methods
for Thioflavin-S staining are well known in the art. All flies are developed
at 27°C.
Thioflavin-S positive cells are not expected to be observed in flies
expressing Tau only.
Thioflavin-S positive cells are expected to be observed in flies expressing
A(342Iowa,
A~42Doton, A~42gtenisha Aa4'2Italian~ or A(342Arctic only. However, the number
of Thioflavin-S-
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CA 02543058 2006-04-20
WO 2005/041650 PCT/US2004/034838
positive cells is expected to be much greater in flies expressing both Tau and
A(342I°Wa,
Aa42putch, A~f2FIemish, A~f2Italian, Or A~42arctic.
Example 2 Screening for a therapeutic agent
1. To screen fox a therapeutic agent effective against Alzheimer's disease,
candidate
agents are administered to a plurality of the A(342I°Wa, A(I42Dutoh,
A(~42~~emish, Aa42itatian, or
A[342A,.°~;~Tau transgenic fly larvae that carry the gmr-GAL4 driver
and the transgenes
UAS:aos- A(342I°Wa, A~42D°tcn, A~42nen,;sh, A.(342ltanan, or
A(342A,.°~;° alone or in combination
with UAS:2rraRTauwt. Candidate agents are microinjected into third instar
transgenic
Drosophila melahogaster~ larvae (three to 5 day old larvae). Larvae are
injected through the
cuticle into the hemolymph with defined amounts of each compound using a
hypodermic
needle of 20 gm internal diameter. Following inj ection, the larvae are placed
into glass vials
for completion of their development. After eclosion, the adult flies are
anesthetized with C02
and visually inspected utilizing a dissecting microscope to assess for the
reversion of the
Dr°osophila eye phenotype as compared to control flies in which a
candidate agent was not
administered. An observed reversion of the A(342I°Wa> A~42Dutch,
A(~42Flemish, A(I42Itanan, or
A[342A,.~t;~ ~Tau transgenic fly eye phenotype towards the phenotype displayed
by the control
grrcr-GAL4 driver strain is indicative of an agent that is useful for the
treatment of
Alzheimer's disease.
2. Screening for memory effect
Pavlovian Learning
Flies are trained by exposure to electroshock (12 pulses at 60 V, duration of
1.5 seconds,
interval of 5 seconds) paired with one odor (benzaldehyde (BA, 4%) or
methylcyclohexanol
(MCH, 10°) for 60 seconds) and subsequent exposure to a second odor
without electroshock.
The odor concentrations are adjusted to assume no preference for flies exposed
simultaneously to the two odors before the training. Immediately after
training, learning is
measured by allowing flies to choose between the two odors used during
training. No
preference between odors results in zero (no learning) performance index (PI).
Avoidance of
the odor previously paired with electroshock is expected to produce a 0 < PI
_< 1.00 (see
Tully, T. and Quinn, W. G., J. Comp. Physiol. A Sens. Neural. Behav. Physiol
., 157:263-
277 (195)).
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