In preparation for animal testing of compounds that have
shown efficacy in in vitro and cell based screens for Huntington’s
disease, the Hereditary Disease Foundation (HDF) convened a workshop in Cardiff,
Wales to consider the optimal approach for animal testing. Mouse models are
needed to test important motor and cognitive dysfunctions that mimic those seen
in humans. Several investigators showed videotapes demonstrating the progression
of symptoms in mice using a variety of assessments that test motor and cognitive
functions. Participants raised concerns about the standardization and
reproducibility of these assessments if they are going to be compared across a
diverse set of laboratories. Their concerns fell into three general areas:
methods, equipment, and background strains of mice. Neuropathologic assessments
were also demonstrated and discussed. Concern was raised about standardization
of immunostaining reagents. While cognitive testing will be extremely important
in the search for treatments, few cognitive assessments have been validated in
mice. Work is ongoing in this area. The group also considered whether there is a
preferred mouse model in which to test potential therapeutics. The R6/2 mouse
develops pathology very early and quickly, allowing more rapid throughput; yet a
more slowly developing model might be more representative of the human disease
and would allow testing of agents in earlier stages of the disease. At the end
of the meeting, a subgroup drew up recommendations for a standard protocol that
could be used across many labs to test potential therapies. These
recommendations demand further discussion and will be presented at the Boston
meeting in August, 2002.
By the end of
2002, scientists are likely to have identified several dozen compounds with
demonstrated efficacy in in vitro and cell-based screens for agents to
combat Huntington’s disease (HD). In preparation for the next step, animal
testing, the Hereditary Disease Foundation (HDF) convened a workshop in July,
2002 to consider the optimal approach to animal testing. The 22 scientists who
attended the meeting in Cardiff, Wales, discussed which models to use as well as
the neuropathologic and behavioral markers that should be assessed.
By the end of the meeting, a set of recommendations was developed for
presentation to other HD researchers, in an attempt to achieve consistency and
reproducibility among different investigators searching for treatments.
The human condition
To begin the meeting, participants were introduced to Richard Evans, a
46-year-old man with HD, and his wife, Jenny. Richard learned at the age of 38
that he carried the expanded allele that had led to his mother’s death at the
age of 56. A few years after having the predictive gene test, Richard began
showing symptoms – problems with memory, irritability, clumsiness, and
difficulty carrying on conversations. In recent years, he has experienced low
mood, worsening memory, lack of concentration, some involuntary movements,
problems with balance, and some choking on food. What bothers him the most, he
said, is the loss of independence. In 1997, Richard became part of a research
effort at Cardiff University investigating neural transplantation as a treatment
for HD. He said the research project “gives you a reason to get up every
day,” despite the hardship of the day-long neuropsychiatric testing he
undergoes every six months.
Blair Leavitt reviewed the three major spheres of dysfunction in HD
patients: motor, cognitive, and psychiatric. While the disease has historically
been defined by the involuntary movements, or chorea, after which it was named
Huntington’s chorea, Leavitt said disruptions in voluntary movements are often
more functionally limiting. The human condition informs people working with
animals about what they need to model, he said. The movement disorder tends to
be more consistently progressive, while the neuropsychologic problems can be
wildly variable and often do not correlate with progression of the illness.
Cognitive problems tend to have a more reliable and stable progression, he said,
but many people have almost no cognitive impairment.
Anne Rosser, the
neurologist who introduced Richard, said that in human subjects, set switching
tasks in which subjects must perform a task under one set of rules and then
switch to another set of rules without knowing what the rules are, are a more
consistent measure of disease progression than the development of chorea. In
Richard’s words, “When they change the rules, I get frustrated.”
noted problems with concentration. Walking requires concentration, he said; as
does speaking to a group. He was able to speak to the workshop participants by
focusing on the one person who was speaking to him directly. Rosser said there
are tests in the neuropsychological battery that assess concentration, such as
the Stroop interference test and Trail Making B test. These tests are sensitive
to progression of HD.
Modeling in the mouse
In mouse models of HD, numerous behavioral tests are used to test
important motor and cognitive dysfunctions that mimic those seen in humans.
Elizabeth M. Simpson noted the importance of finding tasks in mice that
test neural pathways as similar as possible to those pathways that are impaired
in humans. “The closer they come, the more likely you are to get a treatment
that is applicable in humans,” she said.
investigators showed videos of the tests used in their labs and the types of
responses seen in the various mouse models. For example, Jenny Morton showed the
progression of impairments in R6/2 mice between the ages of 6 and 12 weeks. She
used the open field task, which assesses general activity and anxiety; beam
walking, which measures fine coordination and balance capabilities, and a
swimming tank test, which measures swimming speed and assesses other motor
abilities. Simon Brooks demonstrated several other tests including motor tasks
such as the paw reaching and rotarod; as well as the visual cliff avoidance
task; the nine-hole box test which is sensitive to attentional defects caused by
striatal lesions; and the conditional righting, contact righting, and negative
geotaxis reflex tests.
also showed videos demonstrating the progressive features in R6/2 mice. At 15
weeks, when the disease is quite advanced, the mouse is disheveled, has marked
body wasting, bradykinesia, marked ataxia, severe dystonia with sustained
contraction of the forelimbs, and helicoptering of the hind limbs. Videos of
mice at 14, 12, 10, 8, 6, and 4 weeks clearly illustrate how these features
progress. At 4 weeks, there is often no diagnostic abnormality, he said, and the
mice are relatively normal in terms of posturing and foraging. By 6 weeks, mild
symptoms are apparent; and by 8 weeks, severe neuropathology is seen. Steven
Dunnett suggested that Ferrante operationalize his observations so that other
investigators could utilize and report comparable results from a similar
Anton van Dellen
showed videos of open field, rotarod, and cognitive tests of R6/1 mice. He noted
the difficulty of characterizing and quantifying some of the behaviors observed,
such as circling and grooming. Wild-type mice progress through a normal grooming
sequence, he said, but HD mice get distracted and do not progress normally.
Jenny Morton said that grooming behaviors are very complicated and that it has
been reported that HD mice get the order of their grooming pattern mixed up.
‘Circling’ behaviour should also be differentiated from ‘running.’
Though superficially they may look similar, the turning associated with
‘running’ is the result of the space limiting walls, said Elizabeth M.
showed videos comparing the pattern of progressive phenotypic changes in five
different HD mouse models: R6/2, N171-82Q, the YAC-72Q line, the inducible
“tet-off” line and MacDonald’s murine knock-in (Hdh-Q92 and Hdh-Q111)
Sally Ditzler described a 2-minute phenotype assessment based on the
SHIRPA protocol. The assessment
requires close observation over the 2 minute period, with evaluators assigning
quantitative scores to the various measures:
latency on the rotarod, clasping, weight, lifespan, physical appearance (e.g,
tremors and appearance of eyes), body and tail position, grooming behavior, and
activity. A composite score is calculated following the assessment.
Several people raised concerns about the standardization and
reproducibility of these behavioral assessments if they are going to be compared
among a diverse set of investigators and laboratories.
Their concerns fell into three general areas: methods, equipment, and
strains of mice.
Elizabeth M. Simpson noted that since mice are nocturnal, the sensitivity of
certain tests depends on the phase of the day/light cycle. Anton van Dellen said
his tests are all done under a red light at night. Handling and housing also
need to be controlled. Simpson said
her lab singly houses mice for all behavioral work. Michael Levine noted that
when the room that R6/2 mice were kept in was isolated from extrinsic noise, the
incidence of seizures was reduced in the mice. Simpson added that mice are more
sensitive to auditory insult than to handling. While noise in the vivarium may
not be under the control of investigators, it nonetheless needs to be controlled
as much as possible in the testing environment. Tests of learning and memory
such as the water maze may be confounded by the motor disabilities of the mice.
Food deprivation may also confound the results, said Simpson. Gender has not
emerged as a significant factor. Gillian Bates said she always breeds through
the males but tests in the females.
Probably the most universally used motor task involves the rotarod.
The rotarod is easier to quantify than other assessments of motor function such
as the open field task, said van Dellen. Some
investigators use an accelerating speed protocol, while some use a constant
speed protocol, which requires more handling as the mice are placed on the
rotarod repeatedly. Moreover,
people train the mice differently, record measurements differently, give the
mice different rest periods between tests, and present their data and analyze
statistics differently. Susan Browne uses the rotarod to study the rate of motor
decline in 5 different mouse models. She said they can pick up more subtle
differences using the accelerating rotarod and that the results are more
reproducible day to day.
Emma Hockly noted
a further complication with the rotarod: mice learn to grip the rod and ride
around rather than falling off. She overcame this problem by modifying the rod
with inner tubes from bicycle tires, which are smooth rather than grooved. She
also noted that you can not rely on the trip plate to measure the time when the
mice drop off. In her tests of R6/2
mice, she accelerates the rod from 4 rpm to 40 rpm over 600 seconds and does
three trials a day without seeing drop off in the third trial. Assessments are
done on consecutive days: 4 consecutive days at 4 weeks, using data from the
last two days to set the baseline; and on three consecutive days at 8 and 12
weeks, using data from the second and third trials. They also weigh and measure
grip strength using a home-made grip strength apparatus. She said they have not
good experience with commercially available grip strength instruments.
commented that measuring rotarod performance at three different
time points may not give a clear measure of when motor performance starts
to decline or its rate of decline. “Different approaches give different types
of information that may be more applicable to different studies,” she said.
Browne also said that failure to ‘trip the time switch’ should not
present a major problem as the mice must be watched carefully in all studies and
fall time can be easily recorded.
of mice. Various mouse transgenic
models are used among the laboratories represented and each has its benefits
(discussed further below). But even for the same transgene, there will be
genetic variability introduced when the background strains and breeding
approaches differ. Especially when you push the limits, said Elizabeth M.
Simpson, subtle genetic differences may result in significant behavioral
differences. Genetic strain background effects are best defined and studied by
breeding full congenic strains on several different pure genetic backgrounds
(e.g., C57BL/6J, CBA/J, and 129S1/SvImJ (JAX®,
002448)) and then looking for commonalities in these different strains. Simpson
said that because human Huntington features are defined across different genetic
backgrounds, the ideal mouse model will have human-like phenotypic features that
are consistent across different genetic backgrounds.
Robert Ferrante, Blair Leavitt, Michael Levine,
and Gillian Bates showed slides demonstrating the neuropathologic features they
see in R6/2, YAC128 (full length), and 140 CAG repeat knock-in mice.
In R6/2 mice at 21 days of age, Ferrante sees slight separation of the
ventricles, the beginnings of aggregate formation in the cortex with diffuse
staining of long projection neurons in the neostriatum, nuclear aggregation in
the hippocampus, but no immunostaining for huntingtin (htt) in the striatum. At
six weeks, he sees 50% striatal volume loss, immunostaining for htt throughout
the neocortex, and a diffuse pattern of staining in the neostriatum, cortex, and
striatum. Neither Jenny Morton nor Gill Bates have seen the atrophy of
the striatum that Ferrante reported.
In YAC128 mice at 6 months of age, Leavitt showed no
significant volume change or cell loss in the striatum or cortex. By 9 months of
age, however, he sees significant volume change, increased density, and some
loss of cells. At 12 months, ventricular dilation is seen along with a 30%
volume change. While no clear nuclear inclusions are seen, there appears to be
an increase in fine nuclear staining. By 18 months of age (very late in the life
of a YAC128 mouse), nuclear inclusions appear with more aggregates in the cortex
than the striatum.
Mike Levine presented slides of the knock-in models studied in the
laboratory of Marie-Francoise Chesselet. Both behavioral anomalies and
neuropathology, similar to that shown by Robert Ferrante, are seen earlier in
the 140Q mice compared to the 94Q mice. In the 94Q mice, there is only a small
amount of cell loss, but this has not yet been assessed in the 140Q mice.
Behavioral changes follow the same pattern (earlier in the 140Qs), he
said, noting that in both the 94Q and 140Q models, behavioral changes are seen
before the appearance of visible microaggregates.
of immunostaining methods may also be helpful if results from various labs are
to be compared. Many people are using the EM48 polyclonal antibody created by
Xiao-Jiang Li against huntingtin protein. Gill Bates used a different antibody,
a polyclonal antibody (S830) raised in sheep against huntingtin. Her slides,
from frozen sections of the R6/2 hippocampus, 15 microns (approximately one
cell) thick, showed extranuclear aggregates in the CA2 as well as in the
striatum. Jenny Morton said that
Paul Patterson has made a series of 8 monoclonal antibodies (MW1-8) that
recognize different fragments of the exon 1 protein. The EM48 and MW
antibodies are available through the HDF website, and characterization of
the antibodies has been published[i].
The comments of Richard and Jenny Evans highlighted the importance of
finding treatments that improve cognitive functioning, yet few cognitive
assessments have been validated in mice. Even
fewer tests exist that look at the emotional, or affective, phenotype. This is
particularly important for drug testing because the cognitive and affective
phenotype may progress differently from the motor phenotype in response to
different drug treatments, said Jenny Morton.
Connie Atwell noted that most cognitive tasks
require motor abilities that eventually decline in both humans and mouse models.
Tests that might be useful in early stages of the disease may not be useful in
later stages, suggesting that two sets of tests may be required. However, Steve
Dunnett said that in tests such as the forced choice swimming task, mice can
learn to swim from light to dark but have trouble reversing when the light is
switched to the opposite end of the tank. Since ability to switch is the
important variable being measured, slower swimming caused by motor impairments
does not make the test unusable; it only means that the test takes longer to
tests that may be useful include the 9-hole nose poke test, which tests sequence
learning and is sensitive to attentional defects, and prepulse inhibition (PPI),
a test of attention and anxiety that is used to study schizophrenia
abnormalities in cortico-striato-thalamic circuitry.
Dunnett said that while PPI is highly quantifiable, specificity may be a
problem. Elizabeth M. Simpson said she studies affective disturbances and has
several tests established to study aggression in mice.
Kosinski asked whether any cognitive tests will fulfill the demands required for
a test to be useful in drug development: easy to carry out, does not require
much training, easy to monitor, gives reliable results with small variability,
and can be used in both early and late stages of disease. Steven Dunnett said some of the tests are useful at various
stages of the disease, but that different rules drop out at different stages.
More complex rules would be required to identify deficits in early stages of the
One of the goals of this meeting, according to Allan Tobin, was to
assess whether the human condition is represented more accurately in full-length
models as compared to models with a truncated gene. Robert Ferrante noted
that a mouse model that is similar to the human disease would be detrimental in
some ways because of the slowness of the human disease. Ethan Signer, however,
noted that even a slowly developing mouse model may have early features that
could be used as models of the human disease.
“There has to be a very good reason for going to a slower model if
it now appears that that all the pathology and behavior in R6/2 mice parallels
that in humans,” said Tobin. Elizabeth
M. Simpson, however, said that investing in just one model would be poor
investing. Different models should prove valuable for different reasons.
Levine concurred. Pathological changes occur in the R6/2 mouse very
early, even before birth. If this model is used to test treatments, he said, you
have to consider that if you start treatment later than weaning you may have
missed the crucial time period for the treatment to work, and see negative
effects that confound the results. The advantage of slower-developing models is
that you get the same sequence of events but over a protracted time course
during which treatments may be tested. “If the goal is to deal with treatment,
you have to combine the two approaches: a
rapid model to use as a screen and slower models to separate things out.”
Gabrielle Schilling discussed the Borchelt mice she uses, transgenic mice
with the prion promoter (PrP) and 18, 44, or 82 CAG repeats. The behavior of
these mice resembles that of the R6 models. However, the different promoter
results in protein expression in different areas of the brain and no inclusions
are seen in peripheral tissue. Michael Levine discussed work with two different
sets of Aronin/DiFiglia transgenic mice with 18, 46, or 100 CAG repeats. The
behavioral phenotype of these mice is less well characterized, although they
appear similar to the R6/2 except on a longer time scale.
Knock-in mice from Peggy Shelbourne’s lab and inducible strains from Ai
Yamamoto/Rene Hen’s lab were also mentioned as models worthy of consideration
for drug testing. Sally Ditzler’s lab is starting a colony of Detloff 125 CAG
mice, which have expanded repeats in both alleles and show phenotypic changes by
6 months of age. Ethan Signer mentioned that Chris Ross has developed an
inducible full-length mouse model; however, data from this model have not yet
been published and the mice are not yet available. Another full-length model,
produce by Dan Tagle, apparently is not available for use by other labs.
More information about the various mouse models can be found in a recent
paper by Menalled and Chesselet [ii]
and on the HDF website: www.hdfoundation.org/PDF/hdmicetable.pdf.
Several investigators raised concerns about the availability and genetic
stability of several transgenic mouse strains. At one time, said Ethan Signer,
the HDF had a policy that in order to receive support, labs must make their mice
available through The Jackson Labs. However, Michael Levine and Elizabeth M.
Simpson noted that demand must usually meet a certain threshold in order for The
Jackson Labs to breed and disseminate rapidly any particular strain. Moreover,
Robert Ferrante said that his lab had observed phenotypic changes in mice
obtained from The Jackson Labs. Ethan
Signer noted that disturbingly different results have been obtained in different
labs testing R6/2 mice. Some of these differences may be due to handling or
environmental enrichment, he said. However, he encouraged investigators to trade
strains and lines to ensure that the differences are not the result of genetic
changes. He said the High-Q foundation will support such efforts.
Another alternative, said Levine, feasible only for labs that have
breeding capability, is to send breeding pairs of mice to labs who want to use a
certain strain. Ferrante said his lab has a transgene core funded by the NIH to
The Cure Huntington’s Disease Initiative (CHDI) was encouraged to look
into the best way to facilitate more efficient distribution of mouse models.
Recommendations for development trials
During the final session of the workshop, Michael Levine, Gillian Bates,
Jenny Morton, and Steven Dunnett outlined suggested preferred practices for drug
testing in mice, based on the previous discussions. They planned to present
these recommendations at the HDF 2002 meeting to be held in Boston, August 9-11,
There was some discussion about the need for these recommendations. A recent
paper in Science[iv] addressed some of
these same issues. According to Allan Tobin, the HDF plans to contract research
and would want all investigators to follow a standardized protocol. David
Reynolds said that in the pharmaceutical industry, standardized screening
protocols are always used. Connie Atwell added that human clinical trials always
use a standard protocol, and that moving from mouse to human trials would be
smoother and faster if the mouse clinical trial is set up as similar to a human
trial as possible.
Carl Johnson added, “There is going to come a time when there are 100
compounds and we are going to want to know roughly how to prioritize them. So we
will have to compare a compound tested in Blair’s lab with one tested in
Jenny’s lab.” That time, said Allan Tobin, may be as early as December,
Which mouse lines to use? A number of mouse models are
available, each with its own merits. In order to produce results that are
comparable yet inclusive of the various models, the group suggested that each
lab run tests in two types of mice: one with a rapidly developing phenotype (e.g,
R6/2, R6/1, or N171) and one with full length genomic htt and a slowly
developing phenotype (e.g., knock-in or YAC). Susan Browne noted that her lab is already using multiple HD
mouse models to assess the efficacy of therapeutic agents.
What is the optimal genetic background and derivation strategy?
The group recommended avoiding starting with a mixed background and then
inbreeding, as this may result in changing phenotypes that make it difficult to
study the mutation in questions. New mutant strains should be backcrossed onto
different inbred strains (e.g., C57BL/6J, CBA/J, and 129S1/SvImJ [JAX®,
002448]) and maintained as congenic inbred strains. As Elizabeth M. Simpson
proposed, characteristics seen consistently on all background strains would
constitute the most relevant phenotype to human studies. Incipient congenic mice
(i.e., backcrossed for at least 5 generations or the equivalent when using “speed congenics”) can begin to
be bred to a second inbred strain and the defined F1 hybrids used for analysis;
a strategy advocated by Simpson.
of reported variability in results obtained using R6/2 mice, the group
recommended genotyping these mice and reporting repeat size in all publications
and in breeding colonies.
Housing. Housing conditions have been shown to dramatically
affect phenotype. Nigel Wood said
he thought it was much more important to standardize animal husbandry before
standardizing the assessments that are to be used. The group recommended
standardization and control of these factors: access to food and water, single
vs. group housing, environmental enrichment, and light/dark cycles. However, it
was noted that some behavioral tests are not sensitive to the light/dark cycle.
Several strategies were discussed for ensuring that mice receive adequate food
and water, including using a longer drinking spout and putting food on the floor
of the cage.
Pathology. The group recommended that a minimum level of
neuropathology should be reported at the disease stages tested in order to
correlate with behavioral phenotype. Stains should include Nissl stain (cresyl
violet), ubiquitin stain (DAKO antibody), htt (e.g., EM48 antibody), and
possibly GFAP. To ensure further consistency, they recommended analyzing coronal
sections at the level of the mid-striatum.
Behavioral phenotypes. This was the main focus of the
workshop. Recommendations were grouped into four categories: neurological
screening, motor assessments, cognitive tests, and affective tests. For general
neurological screening, an operationalized form of the SHIRPA protocol can be
used for screening new lines and for establishing an overall clinical
description. Short forms, such as was described by Sally Ditzler, are available.
However some investigators reported that in their hands, SHIRPA data lacks the
reliability and power associated with some automated tests. Automated tests
would be better, said David Reynolds, because they reduce variability introduced
by different operators.
Motor assessments should include tests
of activity, gait, reflexes, balance, motor coordination, and muscle strength.
The rotarod test of balance was generally accepted as the most sensitive and
could be standardized in terms of equipment, and protocol (accelerating vs.
constant revolutions). Modifications of the equipment might overcome some of the
problems seen with mice clinging to the rod.
Cognitive assessments should also be included in drug testing protocols,
yet at this time it is unclear which cognitive tests should be included.
Cognitive functions that could be tested include memory and learning or
prefrontal, executive functions. In selecting the appropriate tests, it will be
important to separate out motor from cognitive dysfunction.
There is currently minimal data
available using affective assessments in mice, although these issues are of
paramount importance to people with HD. Tests of motivation, sensitivity to
reward, and emotional responses such as depression, anxiety, and aggression
might be extremely useful in evaluating the effectiveness of a drug if they
could be validated in mice.
Design issues. Standardization
of study design was also recommended. As mentioned earlier, the mouse
environment should be standardized in terms of food and water provision,
environmental enrichment, number of transgenic and non-transgenic mice per cage,
and operator consistency. Time of drug administration and time points to be
tested over the lifetime of the animal should also be standardized.
question was raised about the number of animals in each treatment arm that would
yield acceptable power. Emma Hockly said that in her studies of R6/2 mice using
the rotarod protocol, about 13 mice are needed for an 80% chance of detecting an
improvement of 25%. The power will
vary depending on the test and the mouse model, and will need to be calculated
each time, said Gillian Bates. Michael Levine added that some measures will be
amenable to parametric statistics and some will not.
Browne added that the criteria for “animal dropout” from experimental groups
should be standardized. Moreover, she said that the time of onset of motor
decline and the rate of decline may be more informative than performance in the
last surviving mouse. She is currently working with a statistician to develop
the best possible way of analyzing experiments in the future.
Morton suggested that studies of pharmacokinetics, LD50s, mechanism, clearance
and toxicity be contracted out to a contract research organization (CRO) that
has the resources and expertise in this area.
David Reynolds mentioned that drugs on the market are already known to
have pharmacokinetics good enough that they can be dosed by mouth, be absorbed
from the gut, and get into the brain. Allan Tobin said approved drugs that
showed promise in in vitro and cell-based screens may be the first drugs
that will be tested in animals.
Some final thoughts
The workshop ended with some final thoughts about the wisdom of trying to
standardize protocols across labs. Minka vanBeuzekom floated the idea of a core
facility for testing drugs, with additional validation in the individual
laboratories. Simon Brooks suggested that more progress would be made if
different labs geared up to specialize in one particular area, for example,
working on cognitive tests. Connie Atwell noted that cognitive tests that are
sufficient for going forward with trials do not really exist yet for mouse
models of HD or other neurodegenerative diseases. She said that NINDS will
probably be developing a solicitation along these lines in the next year.
Tobin said the idea is to provide some infrastructure that would allow both
trials and the support of discovery-based research. He added that this network
idea will be either validated or proved faulty over the next year and a half.
People will vote with their feet as to whether they wish to be included
in this multicenter trial or not.
[i] Ko J, Ou S, Patteron PH.
2001. New anti-huntingtin monoclonal antibodies: implications for huntingtin
conformation and its binding properties. Brain Res Bull Oct-Nov 1:56
[ii] Menalled LB and Chesselet
M-F, 2002, Mouse models of Huntington’s disease. Trends in
Pharmacologic Sciences, 23,1: 32-39
[iii] power point slides from
this presentation are available on the HDF website, <http://www.hdfoundation.org>
[iv] Crabbe JC, Wahlsten D,
Dudek BC. 1999. Genetics of mouse behavior: Interactions with laboratory
environment. Science 284:1670-1672.