Neurobiology of Disease 10, 201–210 (2002)
doi:10.1006/nbdi.2002.0511
An Inducible Mouse Model of Late Onset
Tay–Sachs Disease
Mylvaganam Jeyakumar, David Smith, Elena Eliott-Smith,
Mario Cortina-Borja,* Gabriele Reinkensmeier, Terry D. Butters,
Thorsten Lemm, † Konrad Sandhoff, † V. Hugh Perry, ‡
Raymond A. Dwek, and Frances M. Platt 1
Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks
Road, Oxford OX1 3QU, United Kingdom; *Centre for Paediatric Epidemiology and
Biostatistics, Institute of Child Health, University College London, 30 Guilford Street,
London, WCIN 1EH, United Kingdom; †Kekulé-Institut für Organische Chemie und
Biochemie der Rheinischen Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Strasse
1D-53121 Bonn, Germany; and ‡School of Biological Sciences, University of Southampton,
Southampton, SO16 7PX, United Kingdom
Received October 18, 2001; revised April 23, 2002; accepted for publication April 30, 2002
Mouse models of the G M2 gangliosidoses, Tay–Sachs and Sandhoff disease, are null for the hexosaminidase ␣ and ␤ subunits respectively. The Sandhoff (Hexbⴚ/ⴚ) mouse has severe neurological
disease and mimics the human infantile onset variant. However, the Tay–Sachs (Hexaⴚ/ⴚ) mouse
model lacks an overt phenotype as mice can partially bypass the blocked catabolic pathway and
escape disease. We have investigated whether a subset of Tay–Sachs mice develop late onset
disease. We have found that ⬃65% of the mice develop one or more clinical signs of the disease within
their natural life span (n ⴝ 52, P < 0.0001). However, 100% of female mice with repeat breeding
histories developed late onset disease at an earlier age (n ⴝ 21, P < 0.0001) and displayed all clinical
features. Repeat breeding of a large cohort of female Tay–Sachs mice confirmed that pregnancy
induces late onset Tay–Sachs disease. Onset of symptoms correlated with reduced up-regulation of
hexosaminidase B, a component of the bypass pathway. © 2002 Elsevier Science (USA)
Key Words: G M2 gangliosidosis; lysosomal storage disease; glycosphingolipid; hexosaminidase;
neurodegeneration.
(Tay–Sachs disease), while mutations in the ␤ sub-unit
gene causes a deficiency of hexosaminidase A and B
(Sandhoff disease). Tay–Sachs (TS) 2 and Sandhoff (SH)
disease both involve G M2 accumulation in the central
nervous system (CNS), leading to progressive neurodegeneration. They occur at a collective frequency of
1–3:300,000 live births (Neufeld, 1991). However, in
the Ashkenazi Jews carrier frequencies are very high
(1 in 30) compared with the general population (1 in
300).
INTRODUCTION
The G M2 gangliosidoses are severe human diseases
characterised by the storage of G M2 ganglioside within
lysosomes. They arise due to an inherited deficiency of
␤-hexosaminidase. There are two major ␤-hexosaminidase isoenzymes, (hexosaminidase A and B), and a
minor form (hexosaminidase S). The three isoenzymes
differ in their sub-unit composition comprising ␣␤, ␤␤
and ␣␣ subunits, respectively. Mutations in the ␣ subunit cause a deficiency of hexosaminidase A and S
2
Abbreviations used: CNS, central nervous system; LOTS, late
onset Tay-Sachs disease; TS, Tay–Sachs disease; SH, Sandhoff disease; GSL, glycosphingolipid; TLC, thin-layer chromatography;
PAS, periodic acid/Schiff reagent.
1
To whom correspondence and reprint requests should be addressed. Fax: 01865 275216. E-mail: [email protected].
0969-9961/02 $35.00
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201
202
The age of onset is variable and patients are categorised as infantile, juvenile or adult/chronic/late
onset (LO). The infantile onset patients have least
residual enzyme activity leading to rapid storage and
early onset of symptoms, while LO variants have appreciable levels of residual enzyme activity and can
present with symptoms at any stage of adult life.
Juvenile onset patients have intermediate levels of
residual enzyme activity and an intermediate age of
symptom onset.
Although Tay–Sachs disease most commonly presents in infancy there are an increasing number of
adult patients who have been diagnosed with the LO
variant. Absolute numbers are difficult to establish
due to the frequent and often repeat misdiagnosis of
this condition due to its superficial resemblance to for
example multiple sclerosis and muscular dystrophy.
Tay–Sachs disease is generally thought of as a paediatric neurological disorder and so is not often considered when making a diagnosis in the adult patients.
As a consequence very little is known about the pathogenic process in the LO variant and there are currently
no animal models with a chronic progressive course.
Mouse models of TS and SH disease have been
generated. Mice deficient in ␤-hexosaminidase A (TS
mice) are phenotypically normal and have a normal
life span (Yamanaka et al., 1994; Cohen-Tannoudji et
al., 1995; Phaneuf et al., 1996; Sango et al., 1995; Taniike
et al., 1995). They accumulate moderate levels of G M2
ganglioside in the CNS, but do not exhibit the neurological symptoms characteristic of human TS disease.
In contrast, mice deficient in ␤-hexosaminidase B (Hex
B, SH mice) develop severe neurological symptoms
and have a life span of only 4 to 4.5 months. The SH
mice resemble the infantile onset human variant. The
lack of symptoms in the TS mouse is the result of
species differences in the ganglioside degradation
pathway. In man, G M2 ganglioside is degraded primarily by ␤-hexosaminidase A (along with the G M2 activator protein) to yield G M3. The absence of ␤-hexosaminidase A in TS disease blocks the pathway and
results in the accumulation of G M2. In mice, G M2 ganglioside can be degraded by a pathway identical to
human, but also via a second pathway where sialidase
acts first on G M2 to yield G A2, which is then further
degraded by Hex B, which is fully functional in TS
disease (Sango et al., 1995).
One limitation of these two mouse models is that
one is asymptomatic (TS) while the other mimics the
most severe human disease variant (SH). It would be
desirable to have a model that resembles the late onset
variants of these diseases, to better understand patho2002 Elsevier Science (USA)
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Jeyakumar et al.
genesis and to evaluate potential therapies. Late onset
Tay–Sachs disease (LOTS) in humans is a chronic progressive, neurodegenerative disease characterised by
progressive dystonia, spinocerebellar degeneration,
motor neurone disease, and psychosis.
In this study, we have identified sporadic cases of
TS mice with late onset disease. We have developed a
method for inducing LOTS in 100% of female mice
and characterised the disease phenotype. The biochemical basis for the development of LOTS disease in
mice has been investigated.
MATERIALS AND METHODS
Mice
Tay–Sachs mice (Hexa⫺/⫺) (C57BL/6 background)
were provided by Dr. Richard Proia (National Institutes of Health, Bethesda, MD 20892) (Taniike et al.,
1995; Yamanaka et al., 1994). Wild-type Hexa (⫹/⫹)
mice were derived from heterozygous matings.
Repeat Breeding of Female Tay-Sachs Mice
Male and female TS mice (Hexa⫺/⫺) were paired
and continuously cohoused. Litters were recorded and
progeny removed at weaning. On average, each multipregnancy female had four litters prior to being separated from the male. For convenience these will be
termed bred females. The bred females were maintained into old age and monitored for clinical signs.
Gait Analysis
Mice were painted with nontoxic water based paint.
Red was applied to the front paws, blue to the hind
paws, and orange on the abdomen. The mice were
placed on Whatman 3 mm paper and allowed to walk
freely.
Skeletal Staining Method
Skeletons were prepared from LOTS mice and age
matched C57BL/6 controls according to the alazarin
staining protocol of Selby (1987).
General Lipid Extraction and Thin-Layer
Chromatography (TLC)
Glycosphingolipid (GSL) extraction and TLC analysis and quantitation was according to previously
published methods (Jeyakumar et al., 1999).
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Mouse Model of Late Onset Tay-Sachs Disease
␤-Hexosaminidase Assay
Statistical Analysis
Tissues (brain and spinal cord) were harvested,
snap frozen in liquid nitrogen, stored at ⫺70°C and
used within 4 weeks. The enzyme source (tissues) was
thawed, homogenized in 3–5 vol of water (Ultraturax
T25) at 4°C. The homogenate underwent three cycles
of freezing and thawing before the assay. Incubation
mixture (in duplicate) containing 5 ␮l enzyme source
and 50 ␮l of the artificial substrate (either 3 mM
4-MUGlcNAc [specific to Hex A and Hex B] or 0.33
mM 4-MUGlcNAc-6-sulfate [specific to Hex A]) in 100
mM citrate/200 mM sodium phosphate buffer pH 4.5
was incubated at 37°C for 30 min and the reaction
stopped by adding 1 ml 0.5M Na 2CO 3 (pH 10.7). Fluorescence was determined in Perkin Elmer spectrofluorimeter with an excitation wavelength of 365
nm and emission wavelength of 450 nm.
LOTS disease frequency was analysed by Chisquared contingency test. Biochemical data were analysed by Student’s t test. The significance of the agedependent interaction of Hex B and G M2 ganglioside
was analysed using four-parameter logistic model
(Vonesh and Chinchilli, 1997) and S-PLUS 3.4 software
(MathSoft, Seattle). The equation used was
G M2 Activator Protein Detection
Individual mouse brains were homogenised in 1 ml
lysing buffer (PBS, 2% Triton X-100 containing protease inhibitors), spun 15 mins at 13,000 rpm. The
supernatant was retained and a Bradford protein assay performed. Sixty micrograms of total protein was
loaded per lane (15% SDS–PAGE under reducing conditions). The gel was transferred to Immobilon-P western blotting membrane (0.8 mA/cm 2 for 2 h) in a
semidry blotter. The membrane was blocked in 10%
dry milk in PBS/0.1% Tween 20. The primary antiserum (goat anti human G M2 activator protein) (Schroder et al., 1993) was incubated in blocking buffer at
1:10,000 dilution overnight at 4°C and washed 3 times
in PBS. The secondary antibody (anti-goat IgG-HRP,
Vector Laboratories) was used at 1:5000 in PBS/
Tween 20 for 1 h at room temperature and visualised
with ECL detection reagent (Amersham).
Sialidase Assay
Sialidase activity against the artificial substrate
(4-MU NeuAc) was measured in tissue homogenates
according to the method of Marchesini (Marchesini et
al., 1981). For assays using the natural substrate (G M2)
the method of Sandhoff et al was followed (Sandhoff
et al., 1977).
Histology; Periodic acid/Schiff Reagent (PAS) and
TUNEL Staining
PAS and TUNEL staining were according to previously published methods (Jeyakumar et al., 1999).
Y⫽␣⫹
␤⫺␣
1 ⫹ exp
冉
冊
共x ⫺ ␭兲
␪
where x is the G M2 quantity, Y is the Hex B activity, ␣
and ␤ are the lower and upper asymptotes respectively, ␭ is the location parameter and ␪ is a scale
parameter governing the velocity of the ascent.
Change point analysis of G M2 and Hex B age-dependent graphs was performed using a Jackknife procedure followed by Bootstrap analysis (Davison and
Hinkley, 1997) programmed on S-PLUS 3.4 program.
RESULTS
Late Onset Tay-Sachs Mice
The majority of TS mice in the colony were asymptomatic throughout their life span. Routine stock management involved animals being humanely killed
prior to 18 months of age, although a small number of
animals were maintained for their natural life span. It
was observed that among these older mice (18 –24
months) spontaneous LOTS disease occurred in both
sexes (⬃65%, n ⫽ 52, P ⬍ 0.0001).
However, a group of females all of the same age
were observed to become symptomatic significantly
earlier (12–18 months of age). The unique feature of
this group was that 100% of the mice were symptomatic (P ⬍ 0.0001, n ⫽ 21). Clinical signs included severe
progressive hind limb weakness with impaired motor
coordination, balance, and mild ataxia. Forelimbs
were functional while the hind limbs were weak,
rigid, and splayed outwards and extensive muscle
atrophy was evident. A concave spine (lordosis) was
an early sign of symptom onset, as was tremor. This
progressed to abdominal dragging with time. The
mice were unable to right themselves when placed on
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Jeyakumar et al.
ture (Fig. 1A), tremor (Fig. 1B), and abdominal dragging (Fig. 1C) were determined over the life span of
the mice.
Amongst males and non-bred females no symptomatic animals were observed prior to 6 months of age
(Figs. 1A–1C). In bred females a detectable tremor was
noted in 6% of the animals in this age group (P ⫽ 0.23,
Fig. 1B). In the 7- to 12-month age group symptomatic
mice were detected (18% of males (P ⫽ 0.04) and 9% of
nonbred females (P ⫽ 0.16) having one or more symptom) (Figs. 1A–1C). In contrast 48% of the bred females in the 7- to 12-month age range exhibited one or
more symptoms (P ⫽ 0.00023) (Figs. 1A–1C). By 13–18
months, 52% of males had spinal curvature (P ⫽
0.00015), 28% tremor (P ⫽ 0.0096), and 14% had progressed to abdominal dragging (P ⫽ 0.079). The nonbred females had a reduced frequency of spinal curvature relative to the males (22%, P ⫽ 0.028) (Fig. 1A)
but 66% had observable tremor (P ⬍ 0.0001) (Fig. 1B)
with 11% progressing to abdominal dragging (P ⫽
0.12) (Fig. 1C). The bred females however had 100 and
92% of animals with back curvature and tremor respectively (P ⬍ 0.0001) and 50% exhibited abdominal
FIG. 1. Frequency and age of symptom onset. Three disease parameters were scored in each group of mice (n ⫽ 10–50 mice per age
group), spinal curvature (lordosis) (A), tremor (B), and abdominal
dragging (C). Data are mean ⫾ SD of values derived from two
independent systematic sampling analysis of data.
their back when the disease was advanced. Weight
loss became apparent, probably due to impaired feeding and was characteristic of all mice in late stage
disease. Although some mice died prematurely others
lived to greater than 24 months, albeit in a compromised state. The only factor these LOTS mice had in
common was that they had had at least four litters
before 12 months of age (all exbreeding stock). The
age-matched females that had not been bred were all
asymptomatic at the same age. These observations
raised the possibility that the LOTS disease phenotype
could be induced at high frequency at an earlier age
using a breeding strategy.
The colony was therefore expanded and the spontaneous frequency of LOTS disease in males and females was determined. In addition, females which had
repeatedly produced litters in the first six months of
life were followed for the development of disease. The
n values ranged from 10 –50 mice for each age category of mice (Fig. 1). The frequency of spinal curva2002 Elsevier Science (USA)
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FIG. 2. Phenotype of LOTS mice. The appearance of an 8-monthold presymptomatic bred female LOTS (A), an 18-month-old symptomatic bred female LOTS (B) and an 18-month-old bred female
C57BL/6 are shown. The gait of these mice is represented in the
lower panel (D–F). Hind paws were painted blue, front paws red
and the abdomen orange.
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Mouse Model of Late Onset Tay-Sachs Disease
FIG. 3. PAS and TUNEL staining of brain sections. Frozen sections were stained with PAS to detect storage GSL (purple). A, C, E, G, I, and
K are from TS mice (⬃18 month old) and B, D, F, H, J, and L–O are from LOTS mice (⬃18 month old). Arrows indicate the PAS-positive
neuronal inclusions and/or neuronal processes. Abbreviations: S2, secondary somatosensory cortex; L-V, cerebral pyramidal neurones of
middle layer of layer 5; L-VI, cerebral polymorphic layer of layer 6; L-VI a and L-VI b subdivisions of layer 6; LEnt, lateral entorhinal cortex;
CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; Py, pyramidal cell layer of the
hippocampus; GrDG, granular layer of the dentate gyrus; CPu, caudate putamen; Crus 1, Crus 1 lobe cerebellum; DC, dorsal column spinal
cord. (M) Neurons from ventral horn of spinal cord; (N) neurons from dorsal horn of spinal cord; (O) Purkinje cells of the cerebellum. P, Q,
and R are TUNEL stained sections from LOTS mice (P, brain stem; Q, cerebellum; R, spinal cord ventral horn). Bar, 10 ␮m.
dragging (P ⫽ 0.0004), the hallmark of advanced disease (Figs. 1A–1C). By 19 –24 months approximately
70% of males, 62% of the nonbred females, and 100%
of the bred females had developed all three symptoms
(P ⬍ 0.0001). Spinal kyphosis was observed in some
mice (males and females) but was not a consistently
observed feature of the LOTS phenotype.
Functional Tests
The phenotype of a presymptomatic 8-month-old
bred female and a symptomatic 18-month-old bred
female is shown (Figs. 2A and 2B). The bred female
LOTS mice had a pronounced change in gait. A representative trace from a presymptomatic bred female
LOTS mouse (8 months old) is shown (Fig. 2D). The
average distance between each pair of prints is 3– 4
cm, comparable to an 18-month-old C57BL/6 control
mouse (Fig. 2F). No abdominal trace was observed.
However, the trace from an 18 month old bred LOTS
female mouse showed two major differences (Fig. 2E).
First, the hind limbs were never brought forward to be
adjacent to the previous position of the front paw,
instead the foot prints were alternating and only approximately 1–1.5 cm apart. Second, a clear abdominal
trace was present due to abdominal dragging.
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206
Skeletal Examination of LOTS Mice
Examination of the complete skeletons of the LOTS
mice (n ⫽ 4, LOTS, n ⫽ 4 C57BL/6 controls), including
the contour of the vertebral column and articulation
and appearance of individual vertebrae postmortem,
showed no osseous deformities. Given the presence of
degenerative disease of upper and motor neurones
with extensive apoptosis in the ventrolateral columns
of the spinal cord, we attribute the lordotic posture to
the flaccid paralysis of the axial and abdominal muscles.
Histopathology
To visualise the extent and distribution of GSL storage material periodic acid-Schiff (PAS) staining was
carried out on frozen brain sections. In the LOTS mice
the intensity of PAS staining and the number of storage neurones were much more pronounced in comparison to the age-matched TS controls (Fig. 3). The
storage was prominent in cortical areas including motor cortex in particular storage was evident in large
pyramidal neurones in the middle layer of layer-V
(Fig. 3B) and the deep cortical polymorphic layer of
layer-VI (Fig. 3B). The neuronal processes in layer-VI
were also intensely stained (Fig. 3B).
In the entorhinal cortex of the LOTS mice (Fig. 3D),
the storage was far more extensive than that observed
in the TS mice (Fig. 3C), such that it was almost
comparable to storage in Sandhoff mice. In CA3 regions of the hippocampus (Fig. 3F) and caudate putamen (Cpu, Fig. 3H) increased storage was noted in the
LOTS mice relative to age-matched TS controls. In the
spinal cord, many areas such as dorsal column (Fig.
3J), ventral horn (Fig. 3M), dorsal horn (Fig. 3N),
Clarke’s column, corticospinal tract, and spinocerebellar tract (data not shown) showed PAS-positive cells in
LOTS mice but were absent in TS age-matched controls (Fig. 3I).
In the cerebellum of LOTS mice, PAS positive cells
were found in almost all cerebellar lobules, whereas in
the TS age-matched controls the storage was restricted
to only some cerebellar lobules such as the simple
lobule, crus 1 and crus 2 ansiform lobules (Figs. 3K–
3L). In the LOTS mice, prominent storage material was
present in the glial and/or neuronal cells in the granular cell layers (Fig. 3L), and at modest level in the
Purkinje and molecular cell neurones (Figs. 3L and
3O). Very little or no storage was observed in the
comparable areas of cerebellum of TS mice at any age
(Fig. 3K).
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To detect apoptosis, brain and spinal cord sections
from LOTS and TS mice (⬃18 months) were stained
using DNA end-labeling technique (TUNEL). No apoptotic cells were detected in any region of the TS
brain or spinal cord. Apoptotic cells were readily observed in the LOTS mice (Figs. 3P–3R). In keeping
with the impaired neurologic function of the LOTS
mice the large neurones (alpha motor neurons) of the
lateral and ventral horn of the spinal cord stained
positive for apoptosis (Fig. 3R), whereas none were
observed in the comparable areas of spinal cord of the
TS mice (data not shown). All other areas of the grey
matter in the spinal cord including the dorsal horn
stained negative for apoptosis (data not shown). In
addition, several areas of the brain stem and cerebellum stained positive for apoptosis (Figs. 3P and 3Q).
To determine whether the progressive motor defect
in LOTS mice is caused by skeletal muscle denervation, a series of muscle sections (quadriceps) were
stained by haematoxylin and eosin. Representative
light micrographs from control C57BL/6 (Fig. 4A),
Tay–Sachs (Fig. 4B), and LOTS (Figs. 4C– 4E) mice (age
matched) are shown. LOTS mice showed several
pathological hallmarks of muscular atrophy, featuring
irregular (moth-eaten) internal architecture, small angular muscle fibers, and clumps of myonuclei (Figs.
4C– 4E). Furthermore, LOTS mice quadriceps muscles
demonstrated a decrease in both muscle diameter and
muscle weight, compared to age matched TS or
C57BL/6 controls (data not shown).
Age-Dependent Analysis of Total Brain G M2
Storage in TS and LOTS Mice
Brain G M2 storage was measured by quantitative
high performance thin layer chromatography (HPTLC)
(Fig. 5). TS mice stored G M2 in an age-dependent linear
fashion up to 6 – 8 months of age. Storage then decreased slightly before it reached a plateau at around
15 months of age (Fig. 5) (see Table 1 for change point
estimates). Both males and females had similar storage
profiles. LOTS mice exhibited a significantly higher
level of storage both in brain (Fig. 5) and spinal cord
(data not shown) relative to age matched asymptomatic TS mice. In the brain of the LOTS mice storage of
G M2 approximated values predicted had storage been
linear with time (Fig. 5), approximately 132% higher
than control values (P ⬍ 0.0001). The G M2 level in the
spinal cord of LOTS mice was 120% higher than control values (P ⬍ 0.05) (data not shown). G M2 ganglioside was not detectable in the brain of wild type mice
at the TLC loading used (data not shown). No G A2 was
207
Mouse Model of Late Onset Tay-Sachs Disease
detected at the TLC loading used in any of the mice
including the LOTS group (data not shown).
Sialidase, ␤-Hexosaminidase B (Hex B) Enzyme
Activities, and G M2 Activator Levels
To determine the relationship between G M2 accumulation and the catabolic enzymes and activator protein
involved in G M2 degradation, sialidase, G M2 activator
(GM2A), and Hex B were measured. In the asymptomatic TS mice and LOTS mice sialidase activity remained relatively constant at all age points, irrespective of whether an artificial or natural substrate was
used in the assays (data not shown). Furthermore
regional brain analysis failed to detect any differences
in sialidase activity between TS and LOTS mice.
GM2A, as determined by western blotting, showed no
evidence of altered expression of this protein in the
brains of LOTS mice relative to C57BL/6 controls (Fig.
6). Interestingly, this protein was highly elevated in
the brains of Sandhoff mice. The major change observed in the TS mice was that the specific activity of
Hex B rose in a sigmoidal fashion at about 7– 8 months
of age (Fig. 7) (see Table 1 for change point estimates)
to reach a plateau at 141.5 ⫾ 3.2% of normal, at about
20 –23 months of age (Fig. 7). This pattern of agedependent Hex B up-regulation was not observed in
wild type mice. Neither the TS nor the wild type mice
showed any gender specific differences in their Hex B
specific activity profiles throughout their life span
(Fig. 7).
In TS mice the estimated age point of initial Hex B
up-regulation (T Hex-B ⫽ 7.26 ⫾ 0.16 months) and of
deviation from linear G M2 storage (T GM2 ⫽ 7.77 ⫾ 0.18
months) were coincident (Table 1). Furthermore, when
the observed specific Hex B activity was plotted as a
function of G M2 ganglioside storage, a correlation between these two parameters was found in males and
females (Fig. 7, inset). Using a logistic regression
model fit of the data the interaction of the two parameters (G M2 vs Hex B) was statistically significant (P ⬍
0.001) both in males and females. In the low G M2
storage range, the estimated base level of Hex B activity was 27–29 units (P ⬍ 0.0001). When the G M2 level
reached 45 to 48 ␮g per 10 mg dry tissue weight, the
Hex B activity increased significantly (P ⬍ 0.00001)
with the G M2 storage to reach a plateau at 41– 45 units
(P ⬍ 0.00001).
In the LOTS mice, the observed high levels of brain
G M2 (but unchanged sialidase and G M2 activator protein) predicts that the compensatory enzyme (Hex B)
may be at a lower level. In keeping with this predic-
tion, the measured specific activities of Hex B were
significantly lower in LOTS mice (18 –27% reduced,
P ⬍ 0.0001), as compared to the age-matched asymptomatic controls (Fig. 7). This indicated that there was
a failure in the LOTS mice to up-regulate Hex B, which
is presumably normally triggered in response to G M2
ganglioside storage in mice.
DISCUSSION
We have found that over 50 percent of Tay–Sachs
mice develop LOTS disease in old age. This has not
previously been observed because published studies
have involved the analysis of mice prior to the age of
spontaneous symptom onset. Several clinical signs
were observed, including lordosis, which progressed
to abdominal dragging, tremor and hind limb weakness/paralysis. Spontaneous LOTS disease in these
mice was variable in terms of age of clinical onset,
clinical presentation and frequency of mice affected.
The mice were therefore of limited value as a model of
human LOTS. However, one group of LOTS mice
were distinct from the sporadic cases as they became
symptomatic at an earlier age and developed all the
clinical manifestations with very similar kinetics. All
of these females had had at least four litters prior to
one year of age. The absence of this phenotype in the
vast majority of nonbred females at the same age
suggested that pregnancy and/or lactation specific
factors may alter aspects of the sialidase/Hex B mediated bypass pathway, either directly or indirectly.
As a consequence, G M2 storage in the CNS exceeded
the toxic threshold level leading to neuronal dysfunction.
When large numbers of females went through multiple pregnancies and were maintained into old age
100% of the mice developed progressive neurological
disease. Within the virgin female and male groups the
maximal level of symptomatic animals observed was
approximately 60% by 18 –24 months of age, whereas
100% of multiple pregnancy females developed symptoms within this time period. The kinetics of disease
development were different between the groups with
approximately 100% of the LOTS mice having tremor
and spinal curvature by 13–18 months compared to
20 –50% of the virgin females and males. We currently
do not know whether a single pregnancy is sufficient
to induce the phenotype, nor do we know which
pregnancy related change is responsible for induction
of disease. It is possible that hormone administration
will induce LOTS, negating the need for repeated
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208
FIG. 4. Muscle pathology. Haematoxylin and Eosin stained quadriceps from control C57BL/6 mice (A), Tay-Sachs (B), and LOTS
(C–E) mice are shown. All mice were between 22 to 23 months old.
Irregular (moth-eaten) internal architecture (arrowheads in C and
D), clumps of myonuclei (black-arrows in C) and small angular
muscle fibres (white-arrow in E) are present in LOTS and absent in
age matched controls (A and B). Bar, 25 ␮m.
breeding. Studies are currently underway to investigate these possibilities.
It was probable that enzymes or cofactors in the
bypass pathway of G M2 catabolism would be responsible for elevated storage and disease in LOTS. We
therefore measured Hex B, sialidase and G M2 activator
protein levels in the symptomatic females and in nonsymptomatic nonbred age matched female TS mice.
We could find no statistically significant changes in
either GM2A or sialidase in the LOTS mice relative to
the TS mice. It was interesting that GM2A levels are
however highly elevated in Sandhoff mice suggesting
that an increase in the levels of this protein is induced
in these mice in response to high levels of storage of
G M2 and G A2. There is no up-regulation of messenger
RNA encoding GM2A protein in Sandhoff mice suggesting a non-transcriptional control mechanism underlying this observation (Potratz et al., 2000).
Hex B levels were measured over the life span of TS
mice and were found to become elevated at approximately 7 months of age, coincident with the deviation
of G M2 storage from linear (0 –7 months). Up-regulation of Hex B is therefore a probable explanation for
how TS mice escape disease. In humans this pathway
is not significant as sialidase activity is limiting. When
Hex B was measured in the brains of bred female
LOTS mice they had significantly reduced levels of
2002 Elsevier Science (USA)
All rights reserved.
©
Jeyakumar et al.
FIG. 5. Quantitative age-dependent analysis of G M2 storage in
brains of Tay–Sachs mice. G M2 was quantified from mouse brains of
different ages as described in materials and method. Data are
mean ⫾ SD of values derived from duplicate measurements. TS,
Tay–Sachs asymptomatic; LOTS, late onset Tay–Sachs. The extrapolated dotted lines indicate the hypothetical storage profile of Tay–
Sachs mice (- - - and - 䡠 - 䡠 - for females and males, respectively) if
linear storage persisted.
Hex B, intermediate between the age matched controls
and the asymptomatic TS mice. The bred females do
up-regulate Hex B but not to the degree required to
escape disease and have approximately 23% less Hex
B than the TS mice of a comparable age. It is also
possible that pregnancy induces the expression of an
isoenzyme in brain with reduced catalytic capability.
These mouse models may help elucidate the regulation of enzyme expression in the mouse in response to
GSL storage and the physiological/hormonal changes
associated with pregnancy.
Hex B levels were analysed in the symptomatic
male mice and a reduction in Hex B up-regulation was
found (16% lower than asymptomatic age matched TS
mice), which suggests that in male mice there is a
range of levels of Hex B up-regulation and at one
extreme this is inadequate to prevent pathological lev-
FIG. 6. G M2 activator levels determined by western blotting. Brain
samples from control C57BL/6 mice or Sandhoff (SH) and LOTS
mice were analyzed by immunoblotting. Data from two separate
blots are shown. The extreme right hand lane is recombinant GM2A
protein.
209
Mouse Model of Late Onset Tay-Sachs Disease
FIG. 7. ␤-hexosaminidase B (Hex B) activities in mouse brains at
different age points. Animals were sacrificed at differing ages and
their brains were removed for ␤ Hexosaminidase assays (pH 4.5)
with either 4-MUGlcNAc (specific to Hex A and Hex B) or 4-MUGlcNAc-6-sulfate (specific to Hex A) as substrates. Hex B activities of
the wild type mice were calculated by subtracting the Hex A activity
from the total. Data are mean values of duplicate measurements.
Positive error bars are shown. TS, Tay–Sachs; WT, wild type; LOTS,
late onset Tay–Sachs; N, normal asymptomatic. Inset: Derived from
the data in Figs. 5 and 7; a four-parameter logistic model fit using
S-PLUS 3.4 statistics software to estimate the change-points for Hex
B and G M2. The x axis is G M2 (␮g/10 mg dry weight) and the y axis
Hex B activity nmol 4-MU/␮g protein/minute.
els of storage within the animals life span. It also
suggests that subtle changes in Hex B levels can have
profound effects on pathogenesis, in keeping with the
model proposed by Conzelmann and Sandhoff (1983).
The data presented therefore demonstrate a correlation between disease escape and Hex B up-regulation. This suggests a causal link between G M2 accumulation and low Hex B levels. However, as sialidase and
GM2A remain apparently unchanged in TS and LOTS
mice it suggests that there must be some direct catabolism of G M2 by Hex B. The elevation in G M2 in LOTS
mice could then lead to reduced efficiency of flux
through the sialidase pathway (substrate inhibition)
leading to a greater accumulation of G M2. It suggests
that both proposed models (direct catabolism of G M2
by Hex B and the sialidase bypass pathway) (Sango et
al., 1995; Phaneuf et al., 1996) may be operating in the
TS and LOTS mice, albeit to differing degrees.
Reduced Hex B levels in the bred females could
result not only in G M2 storage but also in storage of
G A2, analogous to the situation in Sandhoff mice. However, G A2 storage was never detected in the LOTS
mice. Any G A2 that is produced by sialidase activity, is
probably efficiently cleaved by even low levels of Hex
B since the desialylated form of the glycolipid is a
more favourable substrate for the enzyme (Sandhoff et
al., 1977).
The possibility that isoenzymes of sialidase may be
responsible for the failure to detect alterations in sialidase activity, the use of inappropriate assay conditions, or a requirement for tissue restricted factors for
optimal activity, still exists. An eventual resolution
may require in vivo attempts to measure flux of ganglioside through the pathway in intact cells or tissues
derived from TS and LOTS mice.
The brains of the LOTS mice were analysed to study
the regional distribution of the storage material. The
major difference between the symptomatic and
asymptomatic Tay–Sachs mice was the presence of
increased levels of storage in many regions of the
brain including motor-related regions in the LOTS
mice. For example, in the cerebral motor neurones and
their processes in the cerebral cortex, lateral globus
pallidus (LGP), caudate putamen-striatum (CPu), cerebellar neurones and glial cells. The clinical symptoms
of impaired motor coordination and the progressive
hind limb weakness/paralysis detected in the LOTS
mice was consistent with significant storage in this
portion of the brain.
In man, the brain regions affected by adult onset G M2
storage are primarily the cerebellum and motor neuTABLE 1
Statistical Estimates of Change Points a for G M2 and Hex B (brain)
of TS Mice
Estimated change points
(in months)
Data group b
T GM2
T Hex-B
Accuracy
TS females
TS males
TS females ⫹ males
7.58 ⫾ 0.62
7.15 ⫾ 0.64
7.77 ⫾ 0.18
7.38 ⫾ 0.47
6.51 ⫾ 0.34
7.26 ⫾ 0.16
P ⬍ 10 ⫺5
P ⬍ 10 ⫺5
P ⬍ 10 ⫺5
a
Change point is the age point where G M2 storage started to
deviate from linear (T GM2) or where Hex B is upregulated initially
(T Hex-B). These change points were estimated using a Jack-knife and
Bootstrap technique.
b
Group comparison showed no statistically significant difference
in their estimates for both G M2 and Hex B.
©
2002 Elsevier Science (USA)
All rights reserved.
210
rones. The sensorium and the intellect usually remain
intact for most of the course of the disease, unlike
infantile cases. In contrast to the relatively stereotypic
infantile forms, late-onset G M2 gangliosidoses vary
considerably in their age of onset and clinical signs. In
accordance with the human situation, similar observations are made in mice with late-onset Tay–Sachs disease. This variability in mice as well as in humans may
result from small differences in the enzyme activities
between different cell types and brain regions. Small
differences in the rates of ganglioside biosynthesis in
these neurones might also be important. Assaying
lysosomal enzymes in vitro is always problematic as it
may not fully reflect the events which occur within the
intact lysosome. However as the level of G M2 storage
and Hex B correlate very closely it is probable that the
major factor in murine LOTS is the failure to upregulate Hex B.
This inducible mouse model of LOTS disease will be
a useful tool to explore the regulation of enzymes
involved in GSL catabolism in mice. It will also serve
as a system in which to evaluate therapeutic strategies
for the human late onset G M2 gangliosidoses and to
gain a better understanding of pathogenesis.
ACKNOWLEDGMENTS
We are indebted to Professor Tim Cox (Cambridge, UK) for
examining the skeletons of the LOTS mice and for his helpful
comments on the manuscript. We thank Liz Darley for cutting the
brain sections. F.P. is a Lister Institute Research Fellow.
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