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Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats
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Research Report
Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats Miroslava Hrnkova a , Norbert Zilka a,b , Zuzana Minichova a , Peter Koson a , Michal Novak a,b,⁎ a
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 845 10, Bratislava, Slovakia Axon-Neuroscience GmbH, Rennweg 95b, 1030, Vienna, Austria
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Human truncated tau protein is an active constituent of the neurofibrillary degeneration in
Accepted 20 October 2006
sporadic Alzheimer's disease. We have shown that modified tau protein, when expressed as
Available online 13 December 2006
a transgene in rats, induced AD characteristic tau cascade consisting of tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau
Keywords:
complexes. These pathological changes led to the functional impairment characterized by
Alzheimer's disease
a variety of neurobehavioural symptoms. In the present study we have focused on the
Tau truncation
behavioural alterations induced by transgenic expression of human truncated tau.
Transgenic rat
Transgenic rats underwent a battery of behavioural tests involving cognitive- and
Neurobehavioural assessment
sensorimotor-dependent tasks accompanied with neurological assessment at the age of 4.5, 6 and 9 months. Behavioural examination of these rats showed altered spatial navigation in Morris water maze resulting in less time spent in target quadrant (p < 0.05) and fewer crossings over previous platform position (p < 0.05) during probe trial. Spontaneous locomotor activity and anxiety in open field was not influenced by transgene expression. However beam walking test revealed that transgenic rats developed progressive sensorimotor disturbances related to the age of tested animals. The disturbances were most pronounced at the age of 9 months (p < 0.01). Neurological alterations indicating impaired reflex responses were other added features of behavioural phenotype of this novel transgenic rat. These results allow us to suggest that neurodegeneration, caused by the nonmutated human truncated tau derived from sporadic human AD, result in the neuronal dysfunction consequently leading to the progressive neurobehavioural impairment. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
It has been shown that major constituents of neurofibrillary structures in sporadic form of AD are hyperphosphorylated and truncated tau species (Grundke-Iqbal et al., 1986; Wischik
et al., 1988a,b; Novak et al., 1991, 1993). Truncation of tau was suggested to be a possible seminal event in the pathogenesis of Alzheimer's disease and other tauopathies (Novak, 1994; Binder et al., 2005). Recently, we have shown that rat transgenic expression of human truncated tau, derived from
⁎ Corresponding author: Fax: +421 254 774 276. E-mail address: [email protected] (M. Novak). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.10.085
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sporadic Alzheimer's disease, led to the development of AD tau cascade. The cascade was represented by extensive neurofibrillary pathology satisfying several histopathological and biochemical criteria used for identification of neurofibrillary degeneration in AD including tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau complexes (Zilka et al., 2006). Formation of NFT in transgenic animals passed through several immunohistochemically well-defined maturation stages. First stage was defined by pre-tangle formation, the second stage was characterized by formation of intracellular argyrophilic NFTs and the late developmental stage was represented by the presence of extra-neuronal “ghost” tangles (eNFT). As in humans, neurofibrillary degeneration is characterized by extensive formation of sarcosyl-insoluble tau protein complexes displayed three distinctive stages. The first stage of sarcosyl-insoluble monomer (“one band stage”) represents immature developmental stage. The second stage was characterized by intensive phosphorylation (“stage of shifted monomer”). Subsequently, the shifted phosphorylated monomers led to the development of mature sarcosylinsoluble tau complexes (“stage of tau ladder”). Strikingly, the final stage of sarcosyl-insoluble tau formation correlated with the death of animals expressing transgenic human truncated tau. The life span of heterozygote animals was 10–12 months, while the life expectation of wild type rats was 22–24 months. Thus human truncated tau expression directly influences the life span of rats (Zilka et al., 2006). Presented work describes behavioural consequences of AD tau cascade maturing in transgenic rats and the impact of neurofibrillary degeneration on rat neurobehavioural phenotype. This is the first demonstration showing that human truncated tau, when expressed in rat neurons, had dramatic effect on rat neurobehavioural phenotype.
2.
Results
2.1.
Open field test
Transgenic and control rats showed similar spontaneous motor activity in the open field test (F(1,48) = 1.01; p > 0.05; Fig. 1A). Analysis of age-effect did not reveal a significant difference between 4.5, 6 and 9-month-old rats (F(2,96) = 0.340; p > 0.05) and no significant interaction between measured factors (group; age) was found (F(2,96) = 1.235; p > 0.05). Mean moving velocity of transgenic rats did not differ from agematched controls (F(1,48) = 1.38; p > 0.05; Fig. 1B) and at any age tested (F(2,96) = 0.349; p > 0.05). No interaction between independent variables (factors) was observed (F(2,96) = 1.253; p > 0.05). Analysis of distance moved in the central part of the arena yielded similar results, where transgenic and control groups of all ages tested were not significantly different (group effect F(1,48) = 0.219; p > 0.05; age effect F(2,96) = 2.901; p > 0.05; Fig. 1C). No significant interaction between factors was observed (F(2,96) = 0.441; p > 0.05).
2.1.1.
Morris water maze – navigation to hidden platform
During the 5-day training phase, both groups improved their performance significantly by shortening the escape latency
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(F(4,64) = 17.92; p <0.001), and no significant difference between transgenic rats and controls was observed (F(1,16) = 0.22; p >0.05). No interaction between factors age and day (F(4,64) = 0.2751; p> 0.05) was found (Fig. 2A). Swimming velocity (Fig. 2B) was also measured to compare swimming abilities between transgenic rats and controls. Statistical analysis of measured data did not find a difference in swimming velocity between groups (F(1,16) = 0.001; p >0.05), although it has changed between days (F(4,64) = 18.052; p< 0.001). Interaction between group and testing day factor was not found (F(4,64) =0.988; p >0.05). Transgenic rats and age-matched controls performed the acquisition phase of the Morris water task similarly with no sign of spatial orientation deficit and with no evidence of impaired swimming ability. Later decline in the sensorimotor coordination functions prevented transgenic rats to be tested in Morris water maze.
Fig. 1 – Open field test. Total locomotor activity (A) velocity of movement (B) and distance moved in central part of the arena (C) measured in 4.5, 6 and 9-month-old rats. No significant difference between transgenic rats and controls was observed at any of age tested. Diagrams represent mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 12; nctrl = 8. For 6-month-old rats: nTg = 10; nctrl = 10. For 9-month-old rats: nTg = 8; nctrl = 6.
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In 9-month-old transgenic rats 50% of tested subjects were not able to balance on elevated round cross-sectioned beam. Control rats at this age did not show any signs of sensorimotor coordination impairment and performed the task without coordination problems.
2.3.
Prehensile traction test
In the prehensile traction test transgenic animals showed similar performance to age-matched controls (F(1,62) = 0.616; p > 0.05) and no rapid change of muscle strength during aging was observed (F(2,124) = 0.119; p > 0.05; Fig. 5). Similarly there was no significant difference observed between transgenic hemizygotes and age-matched controls at any of age examined (F(2,124) = 2.746; p > 0.05; Fig. 5).
2.4.
Neurological examination
The most profound deficit observed during neurological examination was impairment of hind limb escape extension
Fig. 2 – Morris water maze test – acquisition phase. Escape latency (A) and swim velocity (B) needed to reach the position of the hidden platform during 5 consecutive training days. No difference in acquisition phase of spatial navigation task between transgenic and control rats was observed. Diagrams represent mean ± SEM of values obtained from transgenic (n = 9) and control (n = 9) group of rats.
2.1.2.
Morris water maze – probe trial
During memory retention testing, all rats displayed spatial learning capabilities and significantly preferred the target quadrant location (p < 0.001, t-test). More exact evaluation of probe trial results showed that percent of time spent in target quadrant zone was significantly lower in transgenic rats than in age-matched controls (p < 0.05, t-test; Fig. 3A). Analysis of number of crossings over the previous platform location revealed significant difference between transgenic rats and controls (p < 0.05, t-test; Fig. 3B) indicating the spatial deficit of transgenic rats.
2.2.
Beam walking test
Two-way ANOVA of escape latency showed that traversing time of transgenic rats significantly increased during aging when tested on round-cross-sectioned beam (F(2,162) = 13.06; p < 0.001) and is related to age of tested animals (Fig. 4). Difference between genotypes was significant (F(1,81) = 15.52; p < 0.001) and a significant interaction between age and genotype was also observed (F(2,162) = 3.38; p < 0.05). Bonferroni's post-hoc test showed significant difference between 9month-old transgenic and corresponding control group of rats (p < 0.01).
Fig. 3 – Morris water maze test – probe trial. The percentage of time spent in four quadrants (A) and number of crossings over the previous platform location (B) during the 60-s probe trial performed on last acquisition day. Transgenic rats spent less time searching in target quadrant location (p < 0.05). Similarly, the number of annulus crossings over the platform location showed that transgenic rats reached the original position of the platform significantly less often than their non-transgenic counterparts (p < 0.05). Asterisks indicate significant difference from corresponding control group: *p < 0.05, two-tailed t-test. Diagrams represent mean values ± SEM of 9 animals per group. Tg – transgenic rat, ctrl – age-matched control.
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3.
Fig. 4 – Beam walking test. Escape latency measured in 4.5, 6 and 9-month-old transgenic and control rats. Transgenic rats had significantly longer escape latency than age-matched controls at 9 months of age. **p < 0.01, Bonferroni's post hoc test. Diagram represents mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 28; nctrl = 21. For 6-month-old rats: nTg = 11; nctrl = 12. For 9-month-old rats: nTg = 8; nctrl = 7.
reflex in 7-month-old transgenic rats. The disturbance was clearly manifested, i.e. when an animal was elevated by the tail and posture of hind limbs was observed. Affected transgenic animals held their hind limbs retracted in clasping position. Impairment of this reflex response was observed in 50% of transgenic rats at the age of 7 months. Further neurological examination involving placing reflex and righting reflex, measured as response to a tactile stimulus applied to a dorsal side of the forelimb and time taken to arrange into prone position, were not affected at that age. Later, animals reached the final clinical stage at the age of 10–12 months, characterized by pronounced neurological impairment. At this stage, animals displayed impairment of several reflexes (hind limb escape extension reflex, placing and righting reflexes), bradykinesia and paraparesis.
2.5.
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Discussion
It is noteworthy that naturally disordered protein tau when truncated is inducing full neurofibrillary tau cascade in transgenic rats similar to that seen in human sporadic AD. Neurofibrillary degeneration characterized by massive formation of sarcosyl-insoluble tau protein complexes passed through several well-defined stages. The present study provides experimental data introducing truncated tau protein as an inducer of neurofibrillary changes leading to the progressive deterioration of neurobehavioural functions including spatial memory retention decline, sensorimotor coordination impairment and alterations in reflex responses (Table 1).
3.1.
Spatial learning and memory
It has been proved that rat possess an excellent spatial memory and uses it to guide its navigational activities (Morris et al., 1982; Compton et al., 1997). A complex behavioural test assessing this type of memory is Morris water maze (MWM) (Morris et al., 1982). In our study, reference memory testing paradigm consisted of 5 blocks of four swimming trials followed with single final probe trial. Transgenic rats showed normal hidden-platform acquisition, however they spent less time in target quadrant during probe trial than age-matched controls. Observed memory retention decline was detected already in 4.5-month-old rats. Further analysis of swimming speeds during the acquisition in MWM and total motor activity in open field allow us to rule out sensorimotor impairment and low motivation as possible contributors of spatial memory deficit. Moreover, concomitant immunohistochemical examination of transgenic rats of the same age revealed presence of hyperphosphorylated tau protein distributed in somatodendritic compartment of numerous pyramidal cells in the cortex and hippocampus. It is important to underline that at this
Histopathological findings
Immunohistochemical examination of adult 4.5-month-old transgenic rats revealed hyperphosphorylated tau protein in the pyramidal neurons when using antibody markers AT8 (Ser202, Thr205). Neuronal labelling of hyperphosphorylated tau was observed in the different brain areas including the cortex and the hippocampal formation (Fig. 6). The late clinical stage was characterized at the histopathological level by the presence of massive neurofibrillary degeneration in the spinal cord and brain stem. Neurofibrillary tangles were morphologically similar to human NFTs and were intensely positive for Gallyas silver stain (Fig. 7A) and immunoreactive with phosphorylation-dependent monoclonal antibody AT8 (Fig. 7B). Another pathological hallmark, axonal damage, appeared in the white matter of the spinal cord and brain stem. Damaged axonal fibers were stained by Gallyas silver method (Fig. 7C) and mAb AT8 (Fig. 7D). Immunohistochemical staining showed the presence of a number of axonal spheroids.
Fig. 5 – Prehensile traction test. Prehensile traction score of transgenic rats and age-matched controls measured in 4.5, 6 and 9 months of age. No significant difference between transgenic rats and controls was observed at any of age tested. Diagram represents mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 11; nctrl = 6. For 6-month-old rats: nTg = 13; nctrl = 10. For 9-month-old rats: nTg = 12; nctrl = 15.
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Fig. 6 – Distribution of the hyperphosphorylated tau protein in the cortical and hippocampal neurons. Immunofluorescence staining of the hyperphosphorylated tau protein in the brain of 4-month-old transgenic rat showed massive AT8 immunoreactivity in cortical (A) and hippocampal (C) pyramidal neurons. No immunoreactivity was observed in the age-matched control rat cortex (B) and hippocampus (D). Scale bar: 100 μm.
stage no irreversible neurofibrillary changes represented by argyrophilic neurofibrillary tangles and mature sarcosylinsoluble tau complexes were detected.
3.2.
Sensorimotor functions
Sensorimotor integrative functions were tested by beam walking test, where round cross-sectioned beam was used as an elevated narrow pathway. It is well documented that tasks involving traversing an elevated narrow beam are giving complex information about rat vestibulomotor and sensorimotor functions and allow more sensitive detection of fine motor deficit (Feeney et al., 1982; Goldstein and Davis, 1990; Zausinger et al., 2000). Prolonged traversing latencies suggested that transgenic rats became impaired in performance of this task at the age of 9 months and above. Results obtained from prehensile traction test showed no difference in muscle strength and agility between transgenic and control rats. Therefore impairment of motor coordination functions was not a consequence of the muscle weakness. Furthermore when taking into account results from open field test it was possible to conclude that longer escape latencies were not caused by increased anxiety and/or decreased motor activity, however rather by apparent motor coordination deficit of transgenic animals. These neurobeha-
vioural alterations correlated well with the presence of argyrophilic and phospho-tau positive neurofibrillary tangles and extensive axonal damage in the brain stem and spinal cord. It appears that functional impairment caused by neurodegeneration consequently leads to the progressive reduction of sensorimotor functions. The present study demonstrates that transgenic expression of human truncated tau in the rat central nervous system leads to altered spatial navigation and progressive decline of sensorimotor functions.
4.
Experimental procedures
4.1.
Subjects
The study was performed on hemizygous Axon transgenic rat males of line 318 expressing truncated tau protein under the control of the mouse Thy-1 promoter. Transgenic protein expression levels were 2- to 5-fold over endogenous tau in the isocortex, hippocampus, diencephalon, brain stem and spinal cord (Zilka et al., 2006). Rats were weaned at the age of 5 weeks and kept in standard plastic cages (4–5 animals per cage of size 555 × 345 × 95 mm) with ad libitum access to food and water. The rat colony and testing rooms were maintained in a
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Fig. 7 – Neurofibrillary lesions and axonal damage. Neurofibrillary lesions in the spinal cord stained with Gallyas silver method (A) and immunolabeled with phospho-dependent mAb AT8 (B). Argyrophilic axonal fibers (C) and tau positive axonal spheroids (D) represent characteristic features of axonal damage in the transgenic rat brain. Large axonal swellings are highlighted by arrow. Scale bar: 50 μm.
12 h:12 h light/dark cycle (lights on at 07:00 a.m.), under controlled temperature (22 ± 2 °C) and humidity. All behavioural tests were conducted during the light phase of the diurnal cycle and are summarised in Table 2. Committee of the Institute of Neuroimmunology (Slovak Academy of Sciences) approved all procedures in accordance with relevant legislation.
4.2.
Apparatus and procedures for behavioural studies
4.2.1.
Open field test
The rat’s motor activity in a novel environment (Prut and Belzung, 2003) was tested in a plastic square-shaped arena with 100 × 100 cm floor and 40 cm high walls. For a single trial, the animal was placed in the central part of the arena
Table 1 – Summary of results observed in transgenic rat expressing human truncated tau Behavioural test Open field test
Parameter measured
Total distance of locomotion Velocity of movement Time spent in central area Morris water maze – hidden Escape latency platform acquisition Swimming speed Morris water maze – probe Time spent in correct quadrant trial Beam walking test Traversing time needed to reach the goal area Prehensile traction test Latency to fall from the rod Neurological examination Righting reflex Placing reflex Hind limb escape extension reflex
Age (months)
Results
4.5, 6, 9
No difference
4.5
No difference
4.5
Less time spent in target quadrant, lower number of annulus crossings over previous platform position Sensorimotor coordination impairment found at 9 months
4.5, 6, 9 4.5, 6, 9 7–10 10–12
No difference Impaired hind-limb escape extension reflex response Impaired placing, righting and hind-limb escape extension reflex response, reduction of body weight
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Table 2 – Behavioural tests and age of tested animals involved in single behavioural task Behavioural test Open field test Morris water maze test Beam walking test Prehensile traction test Neurological examination
Age of tested animals 4.5, 6, 9 months 4.5 months 4.5, 6, 9 months 4.5, 6, 9 months 7 months
and permitted to explore it for 15 min. Between individual subjects the arena was cleaned with water. Total length of the path, distance moved in central part of the arena and moving velocity were measured with the use of the EthoVision system version 3.0 (Noldus, Wageningen, The Netherlands).
4.2.2.
Morris water maze test
For the assessment of rat's reference memory, the Morris water maze test was used (Morris et al., 1982). The apparatus consisted of a black circular pool (180 cm in diameter, 75 cm high) filled with water (22 ± 1 °C) to a depth of 37 cm, virtually divided into two zones (central circle and peripheral belt, separated by a boundary ca. 15–17 cm from the wall). The central zone was subdivided into 4 quadrants (NE, NW, SE, SW) of equal size. The principle of the task is to escape from the water by locating a hidden platform (10 cm in diameter) lying 1 cm below the water surface at the centre of the northeast (NE) quadrant. The maze was located in a separate testing room with a number of intramaze and extramaze visual cues. Two 60-W lights placed by the sides of the maze provided the illumination. The escape swim parameters taken by animals between the starting point and the platform were recorded by the video camera located above the centre of testing arena, which was connected to a computer (placed in the next room), supported with computerized video tracking system (Etho-Vision Noldus 3.0, The Netherlands). The software also made it possible to measure time spent or distance moved in defined quadrants during the probe trial.
4.2.2.1. Hidden platform trials for measuring spatial reference memory. For spatial reference memory procedure, rats underwent four trials a day for 5 consecutive training days (only three training trials were performed on the last training day). During all the days of training, the platform position remained stable at the centre of the NE quadrant, only starting points were randomly changed from trial to trial and from day to day between south, east, west and north position. In each trial, the rat was placed at the circumference of the pool, facing the wall and allowed to swim freely until it found the platform or until 60 s elapsed. If the animal found the platform, it was left there for 30 s; if the animal did not find the platform within a set limit, it was guided onto it. After each testing session, the rat was placed in pre-heated cage to get dry. Escape latency was used as a major parameter measured in this task. Escape latencies were calculated as a mean from all training trials performed each day of test performance.
4.2.2.2. Probe trial.
Probe trial was performed as a single 60s swim on the last acquisition day. Rats were placed into the pool from one constant starting position (west) and allowed to swim for 60 s with no platform present in the maze. The videotracking system recorded the time spent in inner four quadrants of the maze.
4.2.3.
Beam walking test
For measuring rat's sensorimotor integration/coordination, the beam walking test performance (Feeney et al., 1982; Goldstein and Davis, 1990) on a round-shaped wooden beam (3.5 cm in diameter and 200 cm long) elevated 75 cm above the ground was measured. The starting area was illuminated with 60-W light bulb placed very close to the beam to represent an aversive stimulus for tested rat. After habituation in the testing room (10 min), rats underwent two shaping and two testing trials, where escape latency was measured. Escape latency represents traversing time from the starting area to the end of the beam, before reaching a goal box. If the animal was not able to traverse, it would be given a maximum time limit of 20 s. The average of two testing trials was evaluated.
4.2.4.
Prehensile traction test
The prehensile traction test measures muscle strength and equilibrium (Combs and D'Alecy, 1987), when the rat's forepaws are placed on a horizontal rope. In our testing procedure, 0.3-cm-thick and 55-cm-long rope, hanged 50 cm above the floor, was used. The cage filled with sawdust was placed under the rope to avoid injury of tested animal. The performance of rats was scored as follows: 0, hangs on 0 to 2 s; 1, hangs on 3 to 4 s; 2, hangs on 5 s, no third limb up to the rope; and 3, hangs on 5 s and brings hind limb up to the rope.
4.2.5.
Neurological examination
Neurological examination consisted of measurement of basic reflexes including righting reflex, tactile and visual placing reflexes and the hind limb escape extension reflex, as described previously (Irwin, 1968; Rogers et al., 1997).
4.3.
Histopathology and immunohistochemistry
Transgenic rats were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2) and the tissues were postfixed immediately after perfusion for 4 h and then cryoprotected, frozen and cut on cryotome or embedded in paraffin. Histological staining (Gallyas silver method) utilized to demonstrate neurofibrillary pathology in neurons and immunohistochemistry using monoclonal antibody AT8 (Innogenetics, Gent, Belgium), that recognizes phosphorylated tau protein, were performed on 10 μm paraffin sections or 50 μm cryosections. For immunohistochemistry, tissue sections were immunostained using the standard avidin biotin peroxidase method (ABC Elite, Vector Laboratories, Burlingame, CA). Sections were then examined with the Olympus BX 51.
4.4.
Statistical analysis
Statistical analysis was carried out with use of Prism GraphPad Software (Graph Pad Software, Inc., USA). Locomotor activity
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and velocity measured in the open field test, the prehensile traction test and escape latency observed in beam walking test were analyzed with two-way ANOVA (with genotype and age as the main between-subject factors) followed with Bonferroni's post hoc test, if main analysis showed statistical significance. The significance of differences between mean scores during acquisition trial blocks in the Morris water maze was assessed with two-way analysis of variance (ANOVA) for testing days and groups with repeated measures on the day factor. For the standard memory retrieval test (probe trial), two-tailed t-test was used to compare the percentage of time spent searching in target quadrant and for analysis of number of annulus crossings over previous platform location. All values p < 0.05 were considered as statistically significant.
Acknowledgment We wholeheartedly thank to Dr. Jan Bures for critical reading of the manuscript, fruitful suggestions and helpful comments.
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Research Report
Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats Miroslava Hrnkova a , Norbert Zilka a,b , Zuzana Minichova a , Peter Koson a , Michal Novak a,b,⁎ a
Institute of Neuroimmunology, Slovak Academy of Sciences, Dubravska cesta 9, 845 10, Bratislava, Slovakia Axon-Neuroscience GmbH, Rennweg 95b, 1030, Vienna, Austria
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Human truncated tau protein is an active constituent of the neurofibrillary degeneration in
Accepted 20 October 2006
sporadic Alzheimer's disease. We have shown that modified tau protein, when expressed as
Available online 13 December 2006
a transgene in rats, induced AD characteristic tau cascade consisting of tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau
Keywords:
complexes. These pathological changes led to the functional impairment characterized by
Alzheimer's disease
a variety of neurobehavioural symptoms. In the present study we have focused on the
Tau truncation
behavioural alterations induced by transgenic expression of human truncated tau.
Transgenic rat
Transgenic rats underwent a battery of behavioural tests involving cognitive- and
Neurobehavioural assessment
sensorimotor-dependent tasks accompanied with neurological assessment at the age of 4.5, 6 and 9 months. Behavioural examination of these rats showed altered spatial navigation in Morris water maze resulting in less time spent in target quadrant (p < 0.05) and fewer crossings over previous platform position (p < 0.05) during probe trial. Spontaneous locomotor activity and anxiety in open field was not influenced by transgene expression. However beam walking test revealed that transgenic rats developed progressive sensorimotor disturbances related to the age of tested animals. The disturbances were most pronounced at the age of 9 months (p < 0.01). Neurological alterations indicating impaired reflex responses were other added features of behavioural phenotype of this novel transgenic rat. These results allow us to suggest that neurodegeneration, caused by the nonmutated human truncated tau derived from sporadic human AD, result in the neuronal dysfunction consequently leading to the progressive neurobehavioural impairment. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
It has been shown that major constituents of neurofibrillary structures in sporadic form of AD are hyperphosphorylated and truncated tau species (Grundke-Iqbal et al., 1986; Wischik
et al., 1988a,b; Novak et al., 1991, 1993). Truncation of tau was suggested to be a possible seminal event in the pathogenesis of Alzheimer's disease and other tauopathies (Novak, 1994; Binder et al., 2005). Recently, we have shown that rat transgenic expression of human truncated tau, derived from
⁎ Corresponding author: Fax: +421 254 774 276. E-mail address: [email protected] (M. Novak). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.10.085
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sporadic Alzheimer's disease, led to the development of AD tau cascade. The cascade was represented by extensive neurofibrillary pathology satisfying several histopathological and biochemical criteria used for identification of neurofibrillary degeneration in AD including tau hyperphosphorylation, formation of argyrophilic tangles and sarcosyl-insoluble tau complexes (Zilka et al., 2006). Formation of NFT in transgenic animals passed through several immunohistochemically well-defined maturation stages. First stage was defined by pre-tangle formation, the second stage was characterized by formation of intracellular argyrophilic NFTs and the late developmental stage was represented by the presence of extra-neuronal “ghost” tangles (eNFT). As in humans, neurofibrillary degeneration is characterized by extensive formation of sarcosyl-insoluble tau protein complexes displayed three distinctive stages. The first stage of sarcosyl-insoluble monomer (“one band stage”) represents immature developmental stage. The second stage was characterized by intensive phosphorylation (“stage of shifted monomer”). Subsequently, the shifted phosphorylated monomers led to the development of mature sarcosylinsoluble tau complexes (“stage of tau ladder”). Strikingly, the final stage of sarcosyl-insoluble tau formation correlated with the death of animals expressing transgenic human truncated tau. The life span of heterozygote animals was 10–12 months, while the life expectation of wild type rats was 22–24 months. Thus human truncated tau expression directly influences the life span of rats (Zilka et al., 2006). Presented work describes behavioural consequences of AD tau cascade maturing in transgenic rats and the impact of neurofibrillary degeneration on rat neurobehavioural phenotype. This is the first demonstration showing that human truncated tau, when expressed in rat neurons, had dramatic effect on rat neurobehavioural phenotype.
2.
Results
2.1.
Open field test
Transgenic and control rats showed similar spontaneous motor activity in the open field test (F(1,48) = 1.01; p > 0.05; Fig. 1A). Analysis of age-effect did not reveal a significant difference between 4.5, 6 and 9-month-old rats (F(2,96) = 0.340; p > 0.05) and no significant interaction between measured factors (group; age) was found (F(2,96) = 1.235; p > 0.05). Mean moving velocity of transgenic rats did not differ from agematched controls (F(1,48) = 1.38; p > 0.05; Fig. 1B) and at any age tested (F(2,96) = 0.349; p > 0.05). No interaction between independent variables (factors) was observed (F(2,96) = 1.253; p > 0.05). Analysis of distance moved in the central part of the arena yielded similar results, where transgenic and control groups of all ages tested were not significantly different (group effect F(1,48) = 0.219; p > 0.05; age effect F(2,96) = 2.901; p > 0.05; Fig. 1C). No significant interaction between factors was observed (F(2,96) = 0.441; p > 0.05).
2.1.1.
Morris water maze – navigation to hidden platform
During the 5-day training phase, both groups improved their performance significantly by shortening the escape latency
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(F(4,64) = 17.92; p <0.001), and no significant difference between transgenic rats and controls was observed (F(1,16) = 0.22; p >0.05). No interaction between factors age and day (F(4,64) = 0.2751; p> 0.05) was found (Fig. 2A). Swimming velocity (Fig. 2B) was also measured to compare swimming abilities between transgenic rats and controls. Statistical analysis of measured data did not find a difference in swimming velocity between groups (F(1,16) = 0.001; p >0.05), although it has changed between days (F(4,64) = 18.052; p< 0.001). Interaction between group and testing day factor was not found (F(4,64) =0.988; p >0.05). Transgenic rats and age-matched controls performed the acquisition phase of the Morris water task similarly with no sign of spatial orientation deficit and with no evidence of impaired swimming ability. Later decline in the sensorimotor coordination functions prevented transgenic rats to be tested in Morris water maze.
Fig. 1 – Open field test. Total locomotor activity (A) velocity of movement (B) and distance moved in central part of the arena (C) measured in 4.5, 6 and 9-month-old rats. No significant difference between transgenic rats and controls was observed at any of age tested. Diagrams represent mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 12; nctrl = 8. For 6-month-old rats: nTg = 10; nctrl = 10. For 9-month-old rats: nTg = 8; nctrl = 6.
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In 9-month-old transgenic rats 50% of tested subjects were not able to balance on elevated round cross-sectioned beam. Control rats at this age did not show any signs of sensorimotor coordination impairment and performed the task without coordination problems.
2.3.
Prehensile traction test
In the prehensile traction test transgenic animals showed similar performance to age-matched controls (F(1,62) = 0.616; p > 0.05) and no rapid change of muscle strength during aging was observed (F(2,124) = 0.119; p > 0.05; Fig. 5). Similarly there was no significant difference observed between transgenic hemizygotes and age-matched controls at any of age examined (F(2,124) = 2.746; p > 0.05; Fig. 5).
2.4.
Neurological examination
The most profound deficit observed during neurological examination was impairment of hind limb escape extension
Fig. 2 – Morris water maze test – acquisition phase. Escape latency (A) and swim velocity (B) needed to reach the position of the hidden platform during 5 consecutive training days. No difference in acquisition phase of spatial navigation task between transgenic and control rats was observed. Diagrams represent mean ± SEM of values obtained from transgenic (n = 9) and control (n = 9) group of rats.
2.1.2.
Morris water maze – probe trial
During memory retention testing, all rats displayed spatial learning capabilities and significantly preferred the target quadrant location (p < 0.001, t-test). More exact evaluation of probe trial results showed that percent of time spent in target quadrant zone was significantly lower in transgenic rats than in age-matched controls (p < 0.05, t-test; Fig. 3A). Analysis of number of crossings over the previous platform location revealed significant difference between transgenic rats and controls (p < 0.05, t-test; Fig. 3B) indicating the spatial deficit of transgenic rats.
2.2.
Beam walking test
Two-way ANOVA of escape latency showed that traversing time of transgenic rats significantly increased during aging when tested on round-cross-sectioned beam (F(2,162) = 13.06; p < 0.001) and is related to age of tested animals (Fig. 4). Difference between genotypes was significant (F(1,81) = 15.52; p < 0.001) and a significant interaction between age and genotype was also observed (F(2,162) = 3.38; p < 0.05). Bonferroni's post-hoc test showed significant difference between 9month-old transgenic and corresponding control group of rats (p < 0.01).
Fig. 3 – Morris water maze test – probe trial. The percentage of time spent in four quadrants (A) and number of crossings over the previous platform location (B) during the 60-s probe trial performed on last acquisition day. Transgenic rats spent less time searching in target quadrant location (p < 0.05). Similarly, the number of annulus crossings over the platform location showed that transgenic rats reached the original position of the platform significantly less often than their non-transgenic counterparts (p < 0.05). Asterisks indicate significant difference from corresponding control group: *p < 0.05, two-tailed t-test. Diagrams represent mean values ± SEM of 9 animals per group. Tg – transgenic rat, ctrl – age-matched control.
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3.
Fig. 4 – Beam walking test. Escape latency measured in 4.5, 6 and 9-month-old transgenic and control rats. Transgenic rats had significantly longer escape latency than age-matched controls at 9 months of age. **p < 0.01, Bonferroni's post hoc test. Diagram represents mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 28; nctrl = 21. For 6-month-old rats: nTg = 11; nctrl = 12. For 9-month-old rats: nTg = 8; nctrl = 7.
reflex in 7-month-old transgenic rats. The disturbance was clearly manifested, i.e. when an animal was elevated by the tail and posture of hind limbs was observed. Affected transgenic animals held their hind limbs retracted in clasping position. Impairment of this reflex response was observed in 50% of transgenic rats at the age of 7 months. Further neurological examination involving placing reflex and righting reflex, measured as response to a tactile stimulus applied to a dorsal side of the forelimb and time taken to arrange into prone position, were not affected at that age. Later, animals reached the final clinical stage at the age of 10–12 months, characterized by pronounced neurological impairment. At this stage, animals displayed impairment of several reflexes (hind limb escape extension reflex, placing and righting reflexes), bradykinesia and paraparesis.
2.5.
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Discussion
It is noteworthy that naturally disordered protein tau when truncated is inducing full neurofibrillary tau cascade in transgenic rats similar to that seen in human sporadic AD. Neurofibrillary degeneration characterized by massive formation of sarcosyl-insoluble tau protein complexes passed through several well-defined stages. The present study provides experimental data introducing truncated tau protein as an inducer of neurofibrillary changes leading to the progressive deterioration of neurobehavioural functions including spatial memory retention decline, sensorimotor coordination impairment and alterations in reflex responses (Table 1).
3.1.
Spatial learning and memory
It has been proved that rat possess an excellent spatial memory and uses it to guide its navigational activities (Morris et al., 1982; Compton et al., 1997). A complex behavioural test assessing this type of memory is Morris water maze (MWM) (Morris et al., 1982). In our study, reference memory testing paradigm consisted of 5 blocks of four swimming trials followed with single final probe trial. Transgenic rats showed normal hidden-platform acquisition, however they spent less time in target quadrant during probe trial than age-matched controls. Observed memory retention decline was detected already in 4.5-month-old rats. Further analysis of swimming speeds during the acquisition in MWM and total motor activity in open field allow us to rule out sensorimotor impairment and low motivation as possible contributors of spatial memory deficit. Moreover, concomitant immunohistochemical examination of transgenic rats of the same age revealed presence of hyperphosphorylated tau protein distributed in somatodendritic compartment of numerous pyramidal cells in the cortex and hippocampus. It is important to underline that at this
Histopathological findings
Immunohistochemical examination of adult 4.5-month-old transgenic rats revealed hyperphosphorylated tau protein in the pyramidal neurons when using antibody markers AT8 (Ser202, Thr205). Neuronal labelling of hyperphosphorylated tau was observed in the different brain areas including the cortex and the hippocampal formation (Fig. 6). The late clinical stage was characterized at the histopathological level by the presence of massive neurofibrillary degeneration in the spinal cord and brain stem. Neurofibrillary tangles were morphologically similar to human NFTs and were intensely positive for Gallyas silver stain (Fig. 7A) and immunoreactive with phosphorylation-dependent monoclonal antibody AT8 (Fig. 7B). Another pathological hallmark, axonal damage, appeared in the white matter of the spinal cord and brain stem. Damaged axonal fibers were stained by Gallyas silver method (Fig. 7C) and mAb AT8 (Fig. 7D). Immunohistochemical staining showed the presence of a number of axonal spheroids.
Fig. 5 – Prehensile traction test. Prehensile traction score of transgenic rats and age-matched controls measured in 4.5, 6 and 9 months of age. No significant difference between transgenic rats and controls was observed at any of age tested. Diagram represents mean values ± SEM. Tg – transgenic rat, ctrl – age-matched control. For 4.5-month-old rats: nTg = 11; nctrl = 6. For 6-month-old rats: nTg = 13; nctrl = 10. For 9-month-old rats: nTg = 12; nctrl = 15.
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Fig. 6 – Distribution of the hyperphosphorylated tau protein in the cortical and hippocampal neurons. Immunofluorescence staining of the hyperphosphorylated tau protein in the brain of 4-month-old transgenic rat showed massive AT8 immunoreactivity in cortical (A) and hippocampal (C) pyramidal neurons. No immunoreactivity was observed in the age-matched control rat cortex (B) and hippocampus (D). Scale bar: 100 μm.
stage no irreversible neurofibrillary changes represented by argyrophilic neurofibrillary tangles and mature sarcosylinsoluble tau complexes were detected.
3.2.
Sensorimotor functions
Sensorimotor integrative functions were tested by beam walking test, where round cross-sectioned beam was used as an elevated narrow pathway. It is well documented that tasks involving traversing an elevated narrow beam are giving complex information about rat vestibulomotor and sensorimotor functions and allow more sensitive detection of fine motor deficit (Feeney et al., 1982; Goldstein and Davis, 1990; Zausinger et al., 2000). Prolonged traversing latencies suggested that transgenic rats became impaired in performance of this task at the age of 9 months and above. Results obtained from prehensile traction test showed no difference in muscle strength and agility between transgenic and control rats. Therefore impairment of motor coordination functions was not a consequence of the muscle weakness. Furthermore when taking into account results from open field test it was possible to conclude that longer escape latencies were not caused by increased anxiety and/or decreased motor activity, however rather by apparent motor coordination deficit of transgenic animals. These neurobeha-
vioural alterations correlated well with the presence of argyrophilic and phospho-tau positive neurofibrillary tangles and extensive axonal damage in the brain stem and spinal cord. It appears that functional impairment caused by neurodegeneration consequently leads to the progressive reduction of sensorimotor functions. The present study demonstrates that transgenic expression of human truncated tau in the rat central nervous system leads to altered spatial navigation and progressive decline of sensorimotor functions.
4.
Experimental procedures
4.1.
Subjects
The study was performed on hemizygous Axon transgenic rat males of line 318 expressing truncated tau protein under the control of the mouse Thy-1 promoter. Transgenic protein expression levels were 2- to 5-fold over endogenous tau in the isocortex, hippocampus, diencephalon, brain stem and spinal cord (Zilka et al., 2006). Rats were weaned at the age of 5 weeks and kept in standard plastic cages (4–5 animals per cage of size 555 × 345 × 95 mm) with ad libitum access to food and water. The rat colony and testing rooms were maintained in a
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Fig. 7 – Neurofibrillary lesions and axonal damage. Neurofibrillary lesions in the spinal cord stained with Gallyas silver method (A) and immunolabeled with phospho-dependent mAb AT8 (B). Argyrophilic axonal fibers (C) and tau positive axonal spheroids (D) represent characteristic features of axonal damage in the transgenic rat brain. Large axonal swellings are highlighted by arrow. Scale bar: 50 μm.
12 h:12 h light/dark cycle (lights on at 07:00 a.m.), under controlled temperature (22 ± 2 °C) and humidity. All behavioural tests were conducted during the light phase of the diurnal cycle and are summarised in Table 2. Committee of the Institute of Neuroimmunology (Slovak Academy of Sciences) approved all procedures in accordance with relevant legislation.
4.2.
Apparatus and procedures for behavioural studies
4.2.1.
Open field test
The rat’s motor activity in a novel environment (Prut and Belzung, 2003) was tested in a plastic square-shaped arena with 100 × 100 cm floor and 40 cm high walls. For a single trial, the animal was placed in the central part of the arena
Table 1 – Summary of results observed in transgenic rat expressing human truncated tau Behavioural test Open field test
Parameter measured
Total distance of locomotion Velocity of movement Time spent in central area Morris water maze – hidden Escape latency platform acquisition Swimming speed Morris water maze – probe Time spent in correct quadrant trial Beam walking test Traversing time needed to reach the goal area Prehensile traction test Latency to fall from the rod Neurological examination Righting reflex Placing reflex Hind limb escape extension reflex
Age (months)
Results
4.5, 6, 9
No difference
4.5
No difference
4.5
Less time spent in target quadrant, lower number of annulus crossings over previous platform position Sensorimotor coordination impairment found at 9 months
4.5, 6, 9 4.5, 6, 9 7–10 10–12
No difference Impaired hind-limb escape extension reflex response Impaired placing, righting and hind-limb escape extension reflex response, reduction of body weight
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Table 2 – Behavioural tests and age of tested animals involved in single behavioural task Behavioural test Open field test Morris water maze test Beam walking test Prehensile traction test Neurological examination
Age of tested animals 4.5, 6, 9 months 4.5 months 4.5, 6, 9 months 4.5, 6, 9 months 7 months
and permitted to explore it for 15 min. Between individual subjects the arena was cleaned with water. Total length of the path, distance moved in central part of the arena and moving velocity were measured with the use of the EthoVision system version 3.0 (Noldus, Wageningen, The Netherlands).
4.2.2.
Morris water maze test
For the assessment of rat's reference memory, the Morris water maze test was used (Morris et al., 1982). The apparatus consisted of a black circular pool (180 cm in diameter, 75 cm high) filled with water (22 ± 1 °C) to a depth of 37 cm, virtually divided into two zones (central circle and peripheral belt, separated by a boundary ca. 15–17 cm from the wall). The central zone was subdivided into 4 quadrants (NE, NW, SE, SW) of equal size. The principle of the task is to escape from the water by locating a hidden platform (10 cm in diameter) lying 1 cm below the water surface at the centre of the northeast (NE) quadrant. The maze was located in a separate testing room with a number of intramaze and extramaze visual cues. Two 60-W lights placed by the sides of the maze provided the illumination. The escape swim parameters taken by animals between the starting point and the platform were recorded by the video camera located above the centre of testing arena, which was connected to a computer (placed in the next room), supported with computerized video tracking system (Etho-Vision Noldus 3.0, The Netherlands). The software also made it possible to measure time spent or distance moved in defined quadrants during the probe trial.
4.2.2.1. Hidden platform trials for measuring spatial reference memory. For spatial reference memory procedure, rats underwent four trials a day for 5 consecutive training days (only three training trials were performed on the last training day). During all the days of training, the platform position remained stable at the centre of the NE quadrant, only starting points were randomly changed from trial to trial and from day to day between south, east, west and north position. In each trial, the rat was placed at the circumference of the pool, facing the wall and allowed to swim freely until it found the platform or until 60 s elapsed. If the animal found the platform, it was left there for 30 s; if the animal did not find the platform within a set limit, it was guided onto it. After each testing session, the rat was placed in pre-heated cage to get dry. Escape latency was used as a major parameter measured in this task. Escape latencies were calculated as a mean from all training trials performed each day of test performance.
4.2.2.2. Probe trial.
Probe trial was performed as a single 60s swim on the last acquisition day. Rats were placed into the pool from one constant starting position (west) and allowed to swim for 60 s with no platform present in the maze. The videotracking system recorded the time spent in inner four quadrants of the maze.
4.2.3.
Beam walking test
For measuring rat's sensorimotor integration/coordination, the beam walking test performance (Feeney et al., 1982; Goldstein and Davis, 1990) on a round-shaped wooden beam (3.5 cm in diameter and 200 cm long) elevated 75 cm above the ground was measured. The starting area was illuminated with 60-W light bulb placed very close to the beam to represent an aversive stimulus for tested rat. After habituation in the testing room (10 min), rats underwent two shaping and two testing trials, where escape latency was measured. Escape latency represents traversing time from the starting area to the end of the beam, before reaching a goal box. If the animal was not able to traverse, it would be given a maximum time limit of 20 s. The average of two testing trials was evaluated.
4.2.4.
Prehensile traction test
The prehensile traction test measures muscle strength and equilibrium (Combs and D'Alecy, 1987), when the rat's forepaws are placed on a horizontal rope. In our testing procedure, 0.3-cm-thick and 55-cm-long rope, hanged 50 cm above the floor, was used. The cage filled with sawdust was placed under the rope to avoid injury of tested animal. The performance of rats was scored as follows: 0, hangs on 0 to 2 s; 1, hangs on 3 to 4 s; 2, hangs on 5 s, no third limb up to the rope; and 3, hangs on 5 s and brings hind limb up to the rope.
4.2.5.
Neurological examination
Neurological examination consisted of measurement of basic reflexes including righting reflex, tactile and visual placing reflexes and the hind limb escape extension reflex, as described previously (Irwin, 1968; Rogers et al., 1997).
4.3.
Histopathology and immunohistochemistry
Transgenic rats were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.2) and the tissues were postfixed immediately after perfusion for 4 h and then cryoprotected, frozen and cut on cryotome or embedded in paraffin. Histological staining (Gallyas silver method) utilized to demonstrate neurofibrillary pathology in neurons and immunohistochemistry using monoclonal antibody AT8 (Innogenetics, Gent, Belgium), that recognizes phosphorylated tau protein, were performed on 10 μm paraffin sections or 50 μm cryosections. For immunohistochemistry, tissue sections were immunostained using the standard avidin biotin peroxidase method (ABC Elite, Vector Laboratories, Burlingame, CA). Sections were then examined with the Olympus BX 51.
4.4.
Statistical analysis
Statistical analysis was carried out with use of Prism GraphPad Software (Graph Pad Software, Inc., USA). Locomotor activity
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and velocity measured in the open field test, the prehensile traction test and escape latency observed in beam walking test were analyzed with two-way ANOVA (with genotype and age as the main between-subject factors) followed with Bonferroni's post hoc test, if main analysis showed statistical significance. The significance of differences between mean scores during acquisition trial blocks in the Morris water maze was assessed with two-way analysis of variance (ANOVA) for testing days and groups with repeated measures on the day factor. For the standard memory retrieval test (probe trial), two-tailed t-test was used to compare the percentage of time spent searching in target quadrant and for analysis of number of annulus crossings over previous platform location. All values p < 0.05 were considered as statistically significant.
Acknowledgment We wholeheartedly thank to Dr. Jan Bures for critical reading of the manuscript, fruitful suggestions and helpful comments.
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