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Animal Models |
From the Experimental Genetics Group,*
Center for Human
Genetics, Flemish Institute for Biotechnology, Katholieke Universiteit
Leuven, Leuven; the Janssen Research
Foundation,
Beerse; the Department of
Pathology,
University Hospitals Leuven,
Leuven; and the Department of Genetic
Epidemiology,§
Katholieke Universiteit Leuven,
Leuven, Belgium
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, missense and splice-site mutations were detected in the human tau gene in patients with inherited frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).13-19 Most of the exonic mutations located within the microtubule binding repeats rendered the tau protein less functional for microtubule binding and stabilization.20,21 Thus the neurodegeneration appeared to be caused by reduced levels of functional tau protein. On the other hand, mutations in intron 10 and mutations N279K and S305N in exon 10 were shown to induce a preponderance of tau protein isoforms with four microtubule-binding repeats.13,14,17,20,22 Likewise, the silent FTDP-17 mutation L284L, located in the exon 10 splicing silencer sequence, resulted in excess exon 10 inclusion.23 These observations were interpreted to mean that increased levels of normal functional tau protein also could provoke neuronal dysfunction. Two previous studies showed a somatodendritic localization and hyperphosphorylation of the exogenous tau protein, similar to the pretangle changes that precede the neurofibrillary pathology in Alzheimer’s disease, in brain of mice transgenic for human tau protein.24,25 However, neither neuronal dysfunction nor abnormal behavior has been demonstrated thus far in mice overexpressing any human tau isoform.24,25
We have also tested the hypothesis that overproduction of tau protein with four microtubule-binding repeats can constitute a gain of a toxic function in vivo. We have generated transgenic mice that overexpress this tau protein isoform specifically in neurons. We report here that these transgenic mice develop an axonopathy that recapitulates some features of known neurodegenerative diseases, including Alzheimer’s disease and other tauopathies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human Tau40 cDNA, which was deduced from the cDNA clone tau40,26 was ligated in the mouse thy-1 expression cassette.27 A PvuI-NotI restriction fragment was microinjected, and transgenic founders were identified by Southern blotting of StuI-restricted mouse tail-biopt DNA. Routine genotyping of transgenic offspring, bred into the FVB/N genetic background, was performed on tail-biopt DNA by polymerase chain reaction (PCR).
Sensorimotor Tests
The behavioral experiments constitute a transversal study in which transgenic mice from different founder strains were tested at the age of 2–4 months. Five groups of mice were tested: 46 wild-type FVB mice, 31 htau40-1 heterozygous mice, 8 htau40-1 homozygous mice, 17 htau40-5 heterozygous mice, and 12 htau40-5 homozygous mice. The animals were subjected to three sensorimotor tasks designed to assess muscle strength, endurance, coordination, and equilibrium.28
The forced swimming test was performed identically to a probe test in the Morris water maze test, as described before.29 One-way analysis of variance (ANOVA) was used to test for differences in swimming distance between the five groups.
The second sensorimotor task measured the ability of the mouse to walk
along a suspended narrow horizontal rod as an index of psychomotor
integration and equilibrium. Each mouse was placed in the middle of a
50-cm-long aluminum rod of 14-mm diameter. The walking rod held an
escape platform at each end and was positioned 50 cm above a flat
surface. If the mouse succeeded in walking on the rod and/or reaching
one of the escape platforms without falling off, it was scored as 1,
and if the animal fell off, it was scored 0. The contingency
2
test was used for comparison between different groups.
When 2
appeared to be significant, odds ratios and their
two-sided 95% confidence intervals were calculated to determine the
strength of the association.
Finally, the inverted wire mesh grid test referred to the ability to
grasp an elevated horizontal wire grid and to remain suspended for 1
minute. The net was positioned 50 cm above a flat surface and measured
40 cm x 20 cm, with meshes of 0.5 cm x 0.5 cm. If the
animal remained suspended for 1 minute, it was scored 1, and if the
animal dropped off, it was scored 0. The contingency 2
test was used for comparison between different groups. When
2
appeared to be significant, odds ratios and their
two-sided 95% confidence intervals were calculated to determine the
strength of the association.
The statistical analyses were conducted with the SAS 6.12 computer package. All reported P values are two-sided and were considered statistically significant when P < 0.05 (the Bonferroni correction was applied for multiple testing).
Western Blotting
Brain and spinal cord tissue were homogenized in 2 ml and 350 µl, respectively, of buffer containing detergents, proteinase, and phosphatase inhibitors, ie, 0.1 mol/L 2-(N-morpholino)ethanesulfonic acid (pH 6.4), 0.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 5 µg/ml leupeptin, 5 µg/ml pepstatin, 200 µmol/L phenylmethylsulfonyl fluoride, 20 mmol/L NaF, 200 µmol/L sodium orthovanadate, 1 µmol/L okadaic acid, 5 µg/ml soybean trypsin inhibitor, 1% Triton X-100, 1% sodium desoxycholate, and 0.1% sodium dodecyl sulfate. Likewise, two sciatic nerves and 15 spinal ganglia of wild-type and htau40-1 heterozygous transgenic mice were dissected and homogenized in 100 µl and 50 µl homogenization buffer, respectively. After centrifugation (100,000 x g for 30 minutes at 4°C), the supernatant was denatured and reduced before separation on Tris-glycine-buffered polyacrylamide gels (8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose filters as described.29 Protein concentration of cleared homogenates was determined with the Bio-Rad Detergent Compatible protein assay (Bio-Rad Laboratories, Hercules, CA), and equal amounts were loaded.
Because the secondary goat anti-mouse antibody bound on Western blot to proteins of 55 and 67 kd, thereby interfering with the monoclonal anti-tau immunoresponses (especially of AT-8 and AT-180), supernatant of brain and spinal cord homogenates were incubated with immobilized protein-G (Pierce, Rockford, IL) at 4°C for 2.5 hours and purified from mouse IgG by centrifugation (8000 rpm, 5 minutes, 4°C). For the same reason, detection of human tau protein in spinal ganglia and sciatic nerve homogenates was performed with polyclonal antibody B19.
The phosphate-independent antibodies directed to tau protein were monoclonal antibodies HT-7 (Innogenetics, Ghent, Belgium) and Tau-5 (Pharmingen, San Diego, CA) and polyclonal antibody B19 (gift of J. P. Brion, Free University of Brussels, Brussels, Belgium).30 Used monoclonal antibodies directed to phosphorylated tau protein epitopes are listed below.
To dephosphorylate the tau protein before densitometric quantification on Western blot, brain and spinal cord homogenates were diluted in a dephosphorylation buffer (Boehringer Mannheim) containing Escherichia coli alkaline phosphatase (type III; Sigma) at 50 units/ml and incubated for 2 hours at 25°C. Samples to be loaded on the gel were prepared as mentioned above.
Histochemistry, Immunohistochemistry, and Antibodies
For immunohistochemical detection of human tau protein in the htau40 transgenic mice, paraformaldehyde (4% in phosphate-buffered saline), fixed free-floating vibratome slices (40 µm) were incubated with different monoclonal and polyclonal antibodies. Brain and spinal cord sections were incubated with biotin-conjugated secondary antibody, submerged in Strept-ABComplex/horseradish peroxidase, and stained with 3,3'-diaminobenzidine tetrahydrochloride. The phosphate-independent antibodies directed to tau protein were HT-7, the epitope of which has been mapped on human tau between positions 159 and 163; Tau-5, directed to an epitope mapped in the middle of human and murine tau protein; and B19, reactive with the tau protein sequence 154–195.30 Applied antibodies directed to phosphorylated tau protein were AT-8, AT-180, AT-270 (Innogenetics), and PHF-1 (gift of P. Davies, Albert Einstein College of Medicine, New York, NY). Their epitopes have been determined as phosphoSer199 and/or phosphoSer202,31,32 phosphoThr231,33 phosphoThr181,33 and phosphoSer396 and/or phosphoSer404,34 respectively. Antibodies Alz-5035,36 and MC-137 (both gifts of P. Davies) are directed to paired helical filament tau protein. For detection of neurofilament subunit NF-H, antibodies SMI-31, SMI-32 (Affiniti, Nottingham, UK), and NF-200 (Sigma, St. Louis, MO) were applied. NF-M and NF-L were detected by antibodies NF-68 and NF-160 (Sigma), respectively. Antibodies NF-68, NF-160, and NF-200 are phosphate-independent; SMI-31 and SMI-32 are phosphate-dependent. Anti-glial fibrillary acidic protein (anti-GFAP) and ubiquitin were purchased (DAKO A/S, Glostrup, Denmark).
Sections of muscle were submitted to standard hematoxylin/eosin (H&E) staining. Tunel staining was applied to analyze the CNS for apoptotic neurons. Bielschowsky’s silver impregnation and thioflavine-S staining were used to determine the presence of tangle-like structures in neurons of the central nervous system of the transgenic mice.29,38
Quantification of Axonopathy in the CNS
Four wild-type FVB mice, 3 htau40-1 and 3 htau40-5 homozygous mice, and 5 htau40-1 heterozygous mice were transcardially perfused with paraformaldehyde (4% in PBS). Brain and spinal cord were immersion-fixed overnight. Vibratome slices (40 µm) were generated from the right brain hemisphere. Eight cortical slices comprising the hippocampal structures were incubated with monoclonal antibody SMI-32. Immunoreactive axonal dilations with a diameter equal to or larger than the diameter of the nucleus of fifth-layer neocortical pyramidal neurons were counted. Likewise, vibratome sections were made of the mainly thoracal part of the spinal cord, and eight randomly taken thoracal sections were incubated with SMI-32. In parallel, the left hemisphere of each brain and the mainly thoracolumbal part of the spinal cord were embedded in paraffin. Microtome sections (6 µm) of brain and spinal cord were impregnated according to the method of Bielschowsky’s silver staining. The number of argyrophilic axonal dilations was counted in three series of three successive slices. Only cortical slices that comprised the hippocampus were considered in the quantitative analysis of the brain, and only axonal dilations equal to or exceeding the diameter of the nucleus of motor neurons were counted in the spinal cord sections. Three researchers independently performed counting, and the number of dilated axons was averaged for each section of spinal cord or hemisphere. Silver impregnation and SMI-32 immunostaining yielded similar results. The Kruskal-Wallis test was used to evaluate the statistical differences in the average number of axonal dilations between the different groups of transgenic mice.
Quantification of Neurons in the Ventral Horn of the Spinal Cord
Six wild-type FVB mice and six htau40-1 homozygous mice that were 3 months old were transcardially perfused with paraformaldehyde (4% in PBS). Spinal cords were immersion-fixed overnight and embedded in paraffin. Microtome sections (6 µm) of the thoracolumbal region were submitted to standard cresyl violet staining. Composite images from a 3CCD color video camera were collected and assembled with appropriate software (AIS/C 4.0; Imaging Research, St. Catharine’s, ON), and the number of neurons in the right ventral horn in three series of three successive sections was quantitated. Two researchers independently performed counting, and the number of neurons, averaged for each mouse, was statistically analyzed by the Kruskal-Wallis test.
Ultrastructural Analysis
For transmission electron microscopy (TEM), specific regions were excised from 40-µm-thick vibratome sections fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS. For immunogold labeling, samples incubated with AT-8 and Alz-50 after permeabilization with Triton X-100 were further incubated with a secondary antibody conjugated to 1.4-nm gold particles and enhanced with HQ-silver (Nanoprobes, Stony Brook, NY). All samples were postfixed with glutaraldehyde, embedded, and processed for electron microscopy. Samples were additionally postfixed with OsO4 before embedding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice that overexpress the four-repeat human tau protein
isoform with two N-terminal inserts were generated using a recombinant
DNA construct based on the mouse thy-1 gene (Figure 1a)
. Three founder strains were selected,
ie, htau40-1, htau40-2, and htau40-5, which transmitted the transgene
in a stable Mendelian fashion. Transgenic mice from the three lines
expressed the human tau protein exclusively in neurons. Western
blotting of brain (Figure 1b)
and spinal cord homogenates (Figure 1c)
with the monoclonal antibody HT-7 demonstrated the highest expression
of human tau protein in transgenic mice from strains htau40-1 and
htau40-2. Western blotting with monoclonal antibody Tau-5 allowed us to
determine the relative levels of human to mouse tau protein by
densitometric scanning (Figure 1, b and c)
. To obtain accurate
quantification of total tau proteins, the tau isoforms were
dephosphorylated by pretreatment of homogenates with alkaline
phosphatase before application to the gel. The murine tau protein in
brain and spinal cord homogenates in wild-type animals was used as an
internal standard for normalization. In heterozygous mice of strain
htau40-1, the level of human tau protein was about four-fold higher
relative to endogenous mouse tau protein levels, whereas in strain
htau40-5 human tau protein levels were 1.5 times those of murine tau
protein. Human tau protein levels in spinal cord were also quantitated
and estimated to be in the same range (Figure 1d)
.
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. Moreover, axons originating from nerve
cell bodies in the spinal ganglia were immunoreactive for monoclonal
antibody HT-7 in the posterior column of the spinal cord (not shown). Behavior and Motor Problems of htau40 Transgenic Mice
Homozygous htau40-1 and htau40-2 transgenic mice already displayed
at weaning some neurological symptoms. When lifted by the tail,
wild-type FVB mice exhibited an escape reflex by extending their legs,
as opposed to homozygous transgenic mice, which flexed their hind
limbs. Before the transgenic mice were subjected to the standard
cognitive test, ie, the Morris water maze,29
their motor
abilities were examined in a forced swimming test. The swimming speed
of htau40-1 homozygous mice was significantly lower than that of
wild-type, heterozygous and homozygous htau40-5 mice. In 1 minute,
homozygous htau40-1 mice traversed only about 70% of the distance
covered by wild-type littermates (Figure 2b)
. Subsequently, this motor
disturbance, which prevented the mice from being tested in the Morris
water maze, was studied in two additional sensorimotor tasks: rod
walking and inverted grid hanging. This showed that the severity of the
impairment was related to the level of expression of the transgene
(Figure 2)
. Compared to wild-type mice, fewer homozygous htau40-1,
heterozygous htau40-1, and homozygous htau40-5 mice were able to walk
on a rod. In comparison with wild-type mice, they were 90, 13, or 5
times more likely, respectively, to fall off the rod. Significantly
more homozygous htau40-1 mice dropped off the rod, in comparison with
homozygous and heterozygous mice of the strain with lowest expression,
and more heterozygous mice of strain htau40-1 fell than heterozygous
htau40-5 transgenic animals (Figure 2a)
. Likewise, significantly more
homozygous mice of strain htau40-1 lost hold of the inverted wire mesh
grid, in comparison with wild-type and other transgenic mice that lack
or express the transgene to a lower extent (Figure 2c)
. These
observations prove that htau40 transgenic mice displayed a reduced
endurance, a postural instability, a loss of motor coordination and of
maintenance of equilibrium, and a muscular weakness.
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. Histochemical, Immunohistochemical, and Ultrastructural Analysis
The brain and spinal cord of 15 homozygous and heterozygous htau40-1, -2, and -5 transgenic mice, aged between 8 weeks and 8 months, were analyzed histochemically, immunohistochemically, and ultrastructurally to determine the neuropathological cause of their phenotype.
H&E staining did not show striking abnormalities. Bielschowsky’s
silver impregnation did reveal, however, grossly dilated axons in brain
and spinal cord. These dilations had mostly a rounded contour and were
often as large as neighboring neuronal cell bodies. In brain,
dilated axons were mainly present in the neocortex, hippocampus, and
thalamus, but were rare in the subcortical white matter, corpus
callosum, and internal capsule (Figure 3, a and b)
. The proximal location of the dilated
axons in the gray matter suggested a proximal type of axonopathy. In
the spinal cord, the dilated axons were present mostly in the gray
matter, as well as in some white matter fiber tracts. In addition to
the dilated axons, silver staining revealed thickened and irregularly
shaped, argyrophilic dystrophic neurites scattered throughout brain and
spinal cord gray matter (Figure 3c)
. However, staining with silver or
thioflavine-S did not reveal neurofibrillary tangles.
|
. Pyramidal neurons in the cortex and
hippocampus and nerve cells within the thalamus stained strongly, as
did the axonal processes in corpus callosum and the mossy fibers of the
hippocampal granular cells. In the spinal cord, nerve cell bodies and
their processes were stained by these antibodies. A more limited set of
neurons and the dystrophic neurites were strongly reactive with
antibodies Alz-50 and MC-1, which recognize a conformational epitope of
the tau protein present in paired helical filaments20
(Figure 4, b and c)
. Neurons immunoreactive for human tau protein and
axonal dilations also stained with antibodies directed against the
three neurofilament subunits, ie, NF-L (anti-NF-68), NF-M
(anti-NF-160), and NF-H (SMI-31, SMI-32, NF-200) (Figure 4, d–f)
. In
brain, dilated SMI-32 immunoreactive axons were abundant in
neocortex and amygdala. Western blot analyses of brain and spinal cord
homogenates with phosphate-dependent tau antibodies confirmed that
transgenic human tau was phosphorylated at different epitopes. As
different antibodies exhibit different affinities for these epitopes,
quantitation on Western blot was not possible. Nevertheless, the
relative degree of phosphorylation of transgenic human tau varied in
both compartments of the CNS: in spinal cord the AT-8 labeling was
always lower than in brain (Figure 5)
.
|
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. The degree of astrogliosis in the
affected regions of spinal cord and cortex correlated with the
expression level of the human tau transgene as well (results not
shown). GFAP immunostaining revealed activated astrocytes in cortex and
spinal cord of homozygous htau40-1 mice, which were only rarely
observed, if at all, in the other transgenic htau40 strains (Figure 6, a–d)
. In addition, immunohistochemical
staining for ubiquitin labeled some of the dilated axons in cortex and
spinal cord (Figure 6, e and f)
.
|
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. They showed
prominent accumulation of neurofilaments, microtubules, mitochondria,
endoplasmatic reticulum, and vesicles (Figure 7d)
. Some dilated axons
showed signs of degeneration. This varied from axons containing few
degenerating mitochondria to axons with numerous dense and
multivesicular bodies. Often the myelin sheat was thinned, detached, or
completely retracted (Figure 7, b and c)
. Also the thick
dystrophic neurites seen with Bielschowsky’s silver staining
corresponded to dilated axons. Morphologically, they were similar to
the dystrophic axons described in Alzheimer’s disease (AD) and other
neurodegenerative diseases.39
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Ultrastructurally, no filamentous aggregates or tangles were evident,
neither directly by TEM nor after staining with AT-8 or Alz-50 and
gold-labeled secondary antibodies. The latter method resulted in gold
particles projecting on microtubuli or dispersed in the cytoplasm
(Figure 8)
.
|
. Some axons had thinned
myelin sheaths and contained axon-Schwann cell networks, as seen in
chronic neuropathies (Figure 9b)
. The functional impairment of the
damaged axons was also reflected by the skeletal muscle fibers that
they innervate. The quadriceps and the gastrocnemius skeletal muscles
of homozygous htau40-1 transgenic mice showed grouping of atrophic
fibers and fascicular atrophy, diagnostic for neurogenic atrophy
(Figure 9c)
. The degree of muscular atrophy correlated with the degree
of CNS pathology, inasmuch as little muscular atrophy was observed in
heterozygous htau40-1 mice and homozygous htau40-5 mice as opposed to
homozygous htau40-1age-matched animals. The reduction in muscle mass
contributed to a loss of body weight of about 30% in homozygous
htau40-1 mice, even when the mice were only 2 months old. As muscle
atrophy was demonstrated in homozygous htau40-1 mice, the possibility
of neuronal loss in the ventral horn of the spinal cord was examined.
No significant reduction in the number of neurons was recorded. In
wild-type and homozygous htau40-1 mice, the average number of neurons
was 49 ± 13 and 44 ± 12, respectively
(P = 0.6310). Rare chromatolytic neurons,
characterized by loss of Nissl substance and an excentric nucleus, were
observed in the ventral horn of homozygous htau40-1 transgenic mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current observations in htau40 transgenic mice constitute the first in vivo evidence for tau protein-mediated axonal damage. In these transgenic mice, the induced intraneuronal excess of tau protein caused axonopathy, evidenced by proximal axonal dilations with accumulation of neurofilaments, microtubuli, mitochondria, and vesicles, and a Wallerian type of degeneration of distal parts of the axons. Although no neuron loss was established, accumulation of ubiquitinated protein conjugates in some of the dilated axons and astrogliosis both in brain and spinal cord of homozygous htau40-1 transgenic mice reflect onset of neurodegeneration. Moreover, this model proves that excess normal tau protein was sufficient to cause neuronal injury, in the absence of intraneuronal neurofibrillary tangles. The degree of axonopathy, psychomotorical impairment, and muscle atrophy was related to the level of expression of the transgene. During the first 8 months, the longest period of observation at this moment, the pathology in the CNS of homozygous htau40-1 mice progressed only slightly. Detailed analysis of the neuropathology in aging mice up to 2 years will enable us to evaluate the progression of the pathology affecting the CNS of homozygous htau40-1 mice.
A two- to threefold augmentation of tau protein in the brain and spinal cord of homozygous htau40-5 transgenic mice resulted already in the somatodendritic redistribution and ensuing conformational alterations, evident from immunoreactivity with Alz-50 and MC-1. The 3'untranslated region of the tau mRNA is known to target the transcript to the cell body and proximal part of the axon42 but is absent in the Thy1-htau40 construct. Similar signaling sequences in the Thy1 mRNA have not been reported, to our knowledge. Hence, the somatodendritic and axonal localization of the htau40 protein may be determined by specific tau protein transport or by diffusion. In addition, the documented phosphorylation of human tau protein may contribute to its presence in the somatodendritic compartment, which is presumed to be a pretangle stage phenomenon.43 Similar redistribution and conformational alterations have also been observed in other transgenic mice in which the expression of the human tau protein was much lower, at about 10%24 and 14%25 of total tau protein. The absence of tangle-like inclusions in all transgenic models generated so far, either with four-repeat or three-repeat tau protein isoforms, could be explained by expression levels, although additional factors might be required.24,25,44 This situation is not essentially different from the problems experienced in obtaining transgenic mice with amyloid plaques, in which the level of expression has been proved to be essential.45 The analogy can be taken further: transgenic mice with amyloid plaques have indicated that plaques might not be essential for neuronal injury and that they are a late consequence of ongoing neurodegenerative processes.27,29,45
Not reported before are the observed axonal dystrophy and axonal dilations in the current htau transgenic mice. The dilated axons resemble the axonal dilations seen proximal to an axon ligation or axotomy.46 The stasis of axoplasm reflected by accumulation of neurofilaments and organelles is suggestive of a functional axotomy and defective axonal transport. Axonal spheroids, similar to the dilated axons in the htau40 transgenic mice, have been documented in neurological disorders such as amyotrophic lateral sclerosis, in which disturbed axonal transport is evident.47-49 This finding lends support to the hypothesis that an excess of the four-repeat tau protein would saturate binding sites on the microtubules, thus interfering with kinesin-dependent transport as observed in cellular paradigms.41 Since tau protein is considered to be a major component of the short cross-bridges between microtubules,12 overexpression of tau protein may increase these cross-bridges and thereby hinder normal axonal transport. In this respect, we observed binding of the transgenic htau40 protein to isolated neuronal microtubules isolated from htau40-1 transgenic mice, by Western blotting on taxol-stabilized brain homogenates (results not shown). Likewise, the significantly reduced extension of the intermediate filaments caused by the four-repeat tau protein in CHO cells41 might have an in vivo counterpart in the htau40 transgenic mice. The axonal dilations in the CNS of htau40 transgenic mice also stained with antibodies directed against the three neurofilament subunits, indicating that accumulation of neurofilaments in the proximal part of the axon might contribute to deteriorated axonal transport, as has been postulated in neurofilament-overexpressing transgenic mice.49-51 Since the etiology of any tauopathy is unknown, our findings provide a model to gain insight into the pathological processes that are operating in different tauopathies and in tau protein-mediated axonal transport.
Dystrophic neurites, defined as thickened or irregular neuronal processes immunoreactive for tau protein, are a well-known feature of neuropathological disorders and are considered to mark widespread alteration of the neuronal cytoskeleton. In AD, dystrophic axons appear to be prominent and widespread, are particularly abundant in the hippocampal fiber systems originating from the subiculum, CA1, and the entorhinal cortex and may represent one of the main pathological lesions in AD. The morphological, immunohistochemical, and ultrastructural features of the dystrophic axons seen in brain of the htau40 mice resemble those in AD.39,52
The accumulation of ubiquitin-protein conjugates in the axonal dilations53 and the activation of astrocytes confirmed neuronal injury in cortex and spinal cord of transgenic mice. Astrogliosis was not observed in CNS of heterozygous htau40-1 mice that were between 2 and 4 months old, as opposed to homozygous htau40-1 mice of the same age. This suggests that astrocytes became activated subsequent to and likely as a consequence of neuronal injury. The activated astrocytes did not stain with tau protein-specific antibodies and were therefore clearly different from the tau-positive tufted astrocytes, typical for progressive supranuclear palsy.54
The majority of patients suffering from FTDP-17 and other tauopathies develop tau protein deposits in both neurons and glial cells. The mouse thy-1 promoter used by us caused a widespread expression selectively in neurons of the CNS of htau40 transgenic mice. Therefore, these mice model not all but some of the pathological features of FTDP-17 subtypes, which are characterized by an increased level of four-repeat isoforms present in neuronal cytosol or sequestered in inclusions. At the moment four mutations have been described at positions +3, +13, +14, and +16 of the intron after exon 10 of the tau gene. They all cause overexpression of four-repeat isoforms and segregate with neurodegenerative illness.13,14,55 Recently, tau pathology has been reported for familial multiple system tauopathy with presenile dementia (MSTD)14,56 and disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC),13,20,57 caused by the +3 and +14 mutations, respectively. In MSTD, the proven preponderance of the four-repeat tau protein isoform caused axonal swellings in the spinal cord and tangle formation in brain and spinal cord gray matter. In DDPAC, excess of four-repeat tau protein provoked anterior horn pathology and muscle wasting, in the absence of neurofibrillary tangles. Thus, the prominent axonal swellings in the spinal cord, the neurogenic atrophy of muscles, and, in addition, the causal relationship between these pathological characteristics and their genotype make these htau40 transgenic mice interesting animal models that recapitulate features of MSTD and DDPAC. The pathological findings in the CNS of the current transgenic mouse models and in DDPAC patients indicate that increased levels of tau protein or dysregulation of tau protein expression are sufficient to injure neurons in the CNS.
In human brain, the ratio of tau isoforms with four and three repeats (4R/3R) increases from nearly 1 in healthy individuals to 2, 1.8, and 1.6 in patients suffering from DDPAC,20 MSTD (M. G. Spillantini, personal communication), and progressive subcortical gliosis (PSG),55 respectively. The amount of soluble tau remains constant since the level of 3R tau isoforms decreases proportionally. In murine brain, on the contrary, only 4R tau isoforms are expressed during adulthood.58 One might assume that the 4R/3R balance in adult human neurons is more vulnerable to changes relative to the 4R situation in adult mice, requiring an absolute increase in 4R tau isoforms to initiate neuropathology in the later. Homozygous htau40-5 transgenic mice, estimated to express 4R tau protein two to three times more compared to wild-type mice, already exhibit axonopathy and psychomotorical impairments. In descendants of strains htau40-1 and htau40-2 the same neuropathological features are more prominent. Therefore we believe that the level of overexpression obtained in this study is within the pathophysiological range, reminiscent of the relative twofold increase in 4R tau isoforms in some FTDP-17 subtypes.
As each tauopathy strikes selectively specific groups of neurons, further work should be directed at identifying specific biological triggers or genetic factors that lead to a specific pattern of neuronal injury and neuron degeneration, including tangle formation, thereby provoking the corresponding clinical phenotype. In AD the evident candidates are amyloid precursor protein, presenilin, and ApoE4. Others to be considered are suspected tau protein kinases,59-62 excitotoxins,63 ischemia, and resulting radicals64,65 and even sulfated glycosaminoglycans.66-68 These factors can now be tested by incorporation into the current model, which also offers the potential to screen for drugs and to test therapeutic strategies.
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Acknowledgements |
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Footnotes |
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Supported by FWO-Vlaanderen, by the Interuniversity Network for Fundamental Research (IUAP), by the Biotechnology Program of the Flemish Government (IWT/VLAB/COT-008), by NFWO-Lotto, by the Rooms Fund, by the Janssen Research Foundation, and by Leuven Research and Development.
The first three authors contributed equally to this work.
Accepted for publication August 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. EMBO J 1996, 15:1265-1274[Medline]
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D. Muyllaert, D. Terwel, A. Kremer, K. Sennvik, P. Borghgraef, H. Devijver, I. Dewachter, and F. Van Leuven Neurodegeneration and Neuroinflammation in cdk5/p25-Inducible Mice: A Model for Hippocampal Sclerosis and Neocortical Degeneration Am. J. Pathol., February 1, 2008; 172(2): 470 - 485. [Abstract] [Full Text] [PDF]
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M.-M. Mocanu, A. Nissen, K. Eckermann, I. Khlistunova, J. Biernat, D. Drexler, O. Petrova, K. Schonig, H. Bujard, E. Mandelkow, et al. The Potential for -Structure in the Repeat Domain of Tau Protein Determines Aggregation, Synaptic Decay, Neuronal Loss, and Coassembly with Endogenous Tau in Inducible Mouse Models of Tauopathy J. Neurosci., January 16, 2008; 28(3): 737 - 748. [Abstract] [Full Text] [PDF]
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 K. Eckermann, M.-M. Mocanu, I. Khlistunova, J. Biernat, A. Nissen, A. Hofmann, K. Schonig, H. Bujard, A. Haemisch, E. Mandelkow, et al. The beta-Propensity of Tau Determines Aggregation and Synaptic Loss in Inducible Mouse Models of Tauopathy J. Biol. Chem., October 26, 2007; 282(43): 31755 - 31765. [Abstract] [Full Text] [PDF]
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K. Leroy, A. Bretteville, K. Schindowski, E. Gilissen, M. Authelet, R. De Decker, Z. Yilmaz, L. Buee, and J.-P. Brion Early Axonopathy Preceding Neurofibrillary Tangles in Mutant Tau Transgenic Mice Am. J. Pathol., September 1, 2007; 171(3): 976 - 992. [Abstract] [Full Text] [PDF]
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 K. Sennvik, K. Boekhoorn, R. Lasrado, D. Terwel, S. Verhaeghe, H. Korr, C. Schmitz, T. Tomiyama, H. Mori, H. Krugers, et al. Tau-4R suppresses proliferation and promotes neuronal differentiation in the hippocampus of tau knockin/knockout mice FASEB J, July 1, 2007; 21(9): 2149 - 2161. [Abstract] [Full Text] [PDF]
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E. Rockenstein, M. Torrance, A. Adame, M. Mante, P. Bar-on, J. B. Rose, L. Crews, and E. Masliah Neuroprotective Effects of Regulators of the Glycogen Synthase Kinase-3{beta} Signaling Pathway in a Transgenic Model of Alzheimer's Disease Are Associated with Reduced Amyloid Precursor Protein Phosphorylation J. Neurosci., February 21, 2007; 27(8): 1981 - 1991. [Abstract] [Full Text] [PDF]
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T. Vandebroek, D. Terwel, T. Vanhelmont, M. Gysemans, C. Van Haesendonck, Y. Engelborghs, J. Winderickx, and F. Van Leuven Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3beta or cdk5 J. Biol. Chem., September 1, 2006; 281(35): 25388 - 25397. [Abstract] [Full Text] [PDF]
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K. Schindowski, A. Bretteville, K. Leroy, S. Begard, J.-P. Brion, M. Hamdane, and L. Buee Alzheimer's Disease-Like Tau Neuropathology Leads to Memory Deficits and Loss of Functional Synapses in a Novel Mutated Tau Transgenic Mouse without Any Motor Deficits Am. J. Pathol., August 1, 2006; 169(2): 599 - 616. [Abstract] [Full Text] [PDF]
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 B. C. Kraemer, J. K. Burgess, J. H. Chen, J. H. Thomas, and G. D. Schellenberg Molecular pathways that influence human tau-induced pathology in Caenorhabditis elegans Hum. Mol. Genet., May 1, 2006; 15(9): 1483 - 1496. [Abstract] [Full Text] [PDF]
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K. Boekhoorn, D. Terwel, B. Biemans, P. Borghgraef, O. Wiegert, G. J. A. Ramakers, K. de Vos, H. Krugers, T. Tomiyama, H. Mori, et al. Improved long-term potentiation and memory in young tau-P301L transgenic mice before onset of hyperphosphorylation and tauopathy. J. Neurosci., March 29, 2006; 26(13): 3514 - 3523. [Abstract] [Full Text] [PDF]
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G. Amadoro, M. T. Ciotti, M. Costanzi, V. Cestari, P. Calissano, and N. Canu From the Cover: NMDA receptor mediates tau-induced neurotoxicity by calpain and ERK/MAPK activation PNAS, February 21, 2006; 103(8): 2892 - 2897. [Abstract] [Full Text] [PDF]
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M. Ramsden, L. Kotilinek, C. Forster, J. Paulson, E. McGowan, K. SantaCruz, A. Guimaraes, M. Yue, J. Lewis, G. Carlson, et al. Age-Dependent Neurofibrillary Tangle Formation, Neuron Loss, and Memory Impairment in a Mouse Model of Human Tauopathy (P301L) J. Neurosci., November 16, 2005; 25(46): 10637 - 10647. [Abstract] [Full Text] [PDF]
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T. Rodriguez-Martin, M. A. Garcia-Blanco, S. G. Mansfield, A. C. Grover, M. Hutton, Q. Yu, J. Zhou, B. H. Anderton, and J.-M. Gallo Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies PNAS, October 25, 2005; 102(43): 15659 - 15664. [Abstract] [Full Text] [PDF]
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D. Terwel, R. Lasrado, J. Snauwaert, E. Vandeweert, C. Van Haesendonck, P. Borghgraef, and F. Van Leuven Changed Conformation of Mutant Tau-P301L Underlies the Moribund Tauopathy, Absent in Progressive, Nonlethal Axonopathy of Tau-4R/2N Transgenic Mice J. Biol. Chem., February 4, 2005; 280(5): 3963 - 3973. [Abstract] [Full Text] [PDF]
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M. Ikeda, M. Shoji, T. Kawarai, T. Kawarabayashi, E. Matsubara, T. Murakami, A. Sasaki, Y. Tomidokoro, Y. Ikarashi, H. Kuribara, et al. Accumulation of Filamentous Tau in the Cerebral Cortex of Human Tau R406W Transgenic Mice Am. J. Pathol., February 1, 2005; 166(2): 521 - 531. [Abstract] [Full Text] [PDF]
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 G. V. W. Johnson and W. H. Stoothoff Tau phosphorylation in neuronal cell function and dysfunction J. Cell Sci., November 15, 2004; 117(24): 5721 - 5729. [Abstract] [Full Text] [PDF]
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B. Zhang, M. Higuchi, Y. Yoshiyama, T. Ishihara, M. S. Forman, D. Martinez, S. Joyce, J. Q. Trojanowski, and V. M.-Y. Lee Retarded Axonal Transport of R406W Mutant Tau in Transgenic Mice with a Neurodegenerative Tauopathy J. Neurosci., May 12, 2004; 24(19): 4657 - 4667. [Abstract] [Full Text] [PDF]
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Y. Tatebayashi, N. Haque, Y.-C. Tung, K. Iqbal, and I. Grundke-Iqbal Role of tau phosphorylation by glycogen synthase kinase-3{beta} in the regulation of organelle transport J. Cell Sci., May 1, 2004; 117(9): 1653 - 1663. [Abstract] [Full Text] [PDF]
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 J. AVILA, J. J. LUCAS, M. PEREZ, and F. HERNANDEZ Role of Tau Protein in Both Physiological and Pathological Conditions Physiol Rev, April 1, 2004; 84(2): 361 - 384. [Abstract] [Full Text] [PDF]
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T. Ishizawa, N. Sahara, K. Ishiguro, J. Kersh, E. McGowan, J. Lewis, M. Hutton, D. W. Dickson, and S.-H. Yen Co-Localization of Glycogen Synthase Kinase-3 with Neurofibrillary Tangles and Granulovacuolar Degeneration in Transgenic Mice Am. J. Pathol., September 1, 2003; 163(3): 1057 - 1067. [Abstract] [Full Text] [PDF]
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M. Goedert Neurodegenerative tauopathy in the worm PNAS, August 19, 2003; 100(17): 9653 - 9655. [Full Text] [PDF]
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B. C. Kraemer, B. Zhang, J. B. Leverenz, J. H. Thomas, J. Q. Trojanowski, and G. D. Schellenberg From the Cover: Neurodegeneration and defective neurotransmission in a Caenorhabditis elegans model of tauopathy PNAS, August 19, 2003; 100(17): 9980 - 9985. [Abstract] [Full Text] [PDF]
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W.-L. Lin, J. Lewis, S.-H. Yen, M. Hutton, and D. W. Dickson Filamentous Tau in Oligodendrocytes and Astrocytes of Transgenic Mice Expressing the Human Tau Isoform with the P301L Mutation Am. J. Pathol., January 1, 2003; 162(1): 213 - 218. [Abstract] [Full Text] [PDF]
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B. Allen, E. Ingram, M. Takao, M. J. Smith, R. Jakes, K. Virdee, H. Yoshida, M. Holzer, M. Craxton, P. C. Emson, et al. Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein J. Neurosci., November 1, 2002; 22(21): 9340 - 9351. [Abstract] [Full Text] [PDF]
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V. Kunzi, M. Glatzel, M. Y. Nakano, U. F. Greber, F. Van Leuven, and A. Aguzzi Unhampered Prion Neuroinvasion despite Impaired Fast Axonal Transport in Transgenic Mice Overexpressing Four-Repeat Tau J. Neurosci., September 1, 2002; 22(17): 7471 - 7477. [Abstract] [Full Text] [PDF]
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B. Bu, J. Li, P. Davies, and I. Vincent Deregulation of cdk5, Hyperphosphorylation, and Cytoskeletal Pathology in the Niemann-Pick Type C Murine Model J. Neurosci., August 1, 2002; 22(15): 6515 - 6525. [Abstract] [Full Text] [PDF]
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M. Meins, P. Piosik, N. Schaeren-Wiemers, S. Franzoni, E. Troncoso, J. Z. Kiss, C. Brosamle, M. E. Schwab, Z. Molnar, and D. Monard Progressive Neuronal and Motor Dysfunction in Mice Overexpressing the Serine Protease Inhibitor Protease Nexin-1 in Postmitotic Neurons J. Neurosci., November 15, 2001; 21(22): 8830 - 8841. [Abstract] [Full Text] [PDF]
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T. Ishihara, M. Higuchi, B. Zhang, Y. Yoshiyama, M. Hong, J. Q. Trojanowski, and V. M.-Y. Lee Attenuated Neurodegenerative Disease Phenotype in Tau Transgenic Mouse Lacking Neurofilaments J. Neurosci., August 15, 2001; 21(16): 6026 - 6035. [Abstract] [Full Text] [PDF]
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G. F. Hall, V. M.-Y. Lee, G. Lee, and J. Yao Staging of Neurofibrillary Degeneration Caused by Human Tau Overexpression in a Unique Cellular Model of Human Tauopathy Am. J. Pathol., January 1, 2001; 158(1): 235 - 246. [Abstract] [Full Text] [PDF]
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Y. Saito and S. Murayama Expression of tau immunoreactivity in the spinal motor neurons of Alzheimer's disease Neurology, December 12, 2000; 55(11): 1727 - 1730. [Abstract] [Full Text] [PDF]
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I. Tesseur, J. Van Dorpe, K. Bruynseels, F. Bronfman, R. Sciot, A. Van Lommel, and F. Van Leuven Prominent Axonopathy and Disruption of Axonal Transport in Transgenic Mice Expressing Human Apolipoprotein E4 in Neurons of Brain and Spinal Cord Am. J. Pathol., November 1, 2000; 157(5): 1495 - 1510. [Abstract] [Full Text] [PDF]
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L. Conforti, A. Tarlton, T. G. A. Mack, W. Mi, E. A. Buckmaster, D. Wagner, V. H. Perry, and M. P. Coleman A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse PNAS, October 10, 2000; 97(21): 11377 - 11382. [Abstract] [Full Text] [PDF]
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J. Van Dorpe, L. Smeijers, I. Dewachter, D. Nuyens, K. Spittaels, C. Van den Haute, M. Mercken, D. Moechars, I. Laenen, C. Kuiperi, et al. Prominent Cerebral Amyloid Angiopathy in Transgenic Mice Overexpressing the London Mutant of Human APP in Neurons Am. J. Pathol., October 1, 2000; 157(4): 1283 - 1298. [Abstract] [Full Text] [PDF]
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P. Heutink Untangling tau-related dementia Hum. Mol. Genet., April 1, 2000; 9(6): 979 - 986. [Abstract] [Full Text] [PDF]
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I. Tesseur, J. Van Dorpe, K. Spittaels, C. Van den Haute, D. Moechars, and F. Van Leuven Expression of Human Apolipoprotein E4 in Neurons Causes Hyperphosphorylation of Protein Tau in the Brains of Transgenic Mice Am. J. Pathol., March 1, 2000; 156(3): 951 - 964. [Abstract] [Full Text] [PDF]
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I. Grundke-Iqbal and K. Iqbal Tau Pathology Generated by Overexpression of Tau Am. J. Pathol., December 1, 1999; 155(6): 1781 - 1785. [Full Text] [PDF]
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K. Spittaels, C. Van den Haute, J. Van Dorpe, H. Geerts, M. Mercken, K. Bruynseels, R. Lasrado, K. Vandezande, I. Laenen, T. Boon, et al. Glycogen Synthase Kinase-3beta Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice J. Biol. Chem., December 22, 2000; 275(52): 41340 - 41349. [Abstract] [Full Text] [PDF]
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J. Gotz, F. Chen, R. Barmettler, and R. M. Nitsch Tau Filament Formation in Transgenic Mice Expressing P301L Tau J. Biol. Chem., January 5, 2001; 276(1): 529 - 534. [Abstract] [Full Text] [PDF]
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