Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L)

Abstract

Here, we describe the generation of a novel transgenic mouse model of human tauopathy. The rTg(tau(P301L))4510 mouse expresses the P301L mutation in tau (4R0N) associated with frontotemporal dementia and parkinsonism linked to chromosome 17. Transgene expression was driven by a forebrain-specific Ca(2+) calmodulin kinase II promoter system resulting in high levels of expression in the hippocampus and neocortex. Importantly, transgene expression in this model is induced via the tetracycline-operon responsive element and is suppressed after treatment with doxycycline. Continued transgene expression in rTg(tau(P301L))4510 mice results in age-dependent development of many salient characteristics of hereditary human dementia. From an early age, immunohistochemical studies demonstrated abnormal biochemical processing of tau and the presence of pathological conformation- and phosphorylation-dependent epitopes. Neurofibrillary tangle (NFT) pathology was first observed in the neocortex and progressed into the hippocampus and limbic structures with increasing age. Consistent with the formation of NFTs, immunoblots indicated an age-dependent transition of accumulating tau species from Sarkosyl soluble 55 kDa to insoluble hyperphosphorylated 64 kDa. Ultrastructural analysis revealed the presence of straight tau filaments. Furthermore, the effects of tau(P301L) expression on spatial reference memory were longitudinally tested using the Morris water maze. Compared with nontransgenic age-matched control littermates, rTg(tau(P301L))4510 mice developed significant cognitive impairments from 4 months of age. Memory deficits were accompanied by gross forebrain atrophy and a prominent loss of neurons, most strikingly in hippocampal subdivision CA1. Collectively, these data describe a novel transgenic mouse that closely mimics human tauopathy and may represent an important model for the future study of tau-related neurodegenerative disease.

Figure 1.

Generation and characterization of rTg(tauP301L)4510 mice. A, Diagrammatic representation of activator and responder transgenes in bigenic mice. B, Highest levels of tauP301L expression were achieved in mice harboring the lowest number of responder transgene copies. C, Specific transgene expression in the forebrain was confirmed by in situ hybridization in 2- and 4-month-old rTg(tauP301L)4510 mice. Emulsion-dipped slides revealed silver grains clustered over individual neurons, of tau-positive mice only, indicating expression was restricted to neuronal cells types and that the majority of neuronal cells in the hippocampus and cortex expressed transgenic transcripts. D, Composite images (16 images at ×20) of emulsion-dipped slides from 4-month-old tau-positive and tau-negative mice. E, Biochemical studies confirmed high expression of tauP301L in the hippocampus and cortex and no detectable expression in the spinal cord of 2.5-month-old mice. F, Western blot analysis of postnatal forebrain indicated the earliest expression of transgenic tauP301L in P7 mice. Mouse and human tau were detected with tau-5 antibody (Biosource), and human-specific tau was probed for with T14 (Zymed). To demonstrate the high levels of transgene expression, tau-5 immunoblots were loaded with 3 μg of tau-positive forebrain extract compared with 30 μg of tau-negative lysate. T14 and α-tubulin blots were run in parallel after equal loading of 3 μg of protein across all lanes. neg, Negative; pos, positive; Tg, transgenic;α-Tub,α-tubulin; Olf, olfactory bulb; Ctx, cortex; Hpp, hippocampus; Cbm, cerebellum; BnSt, brainstem; Spn, Spinal cord.

Figure 2.

Accumulation of pathological tau species in rTg(tau_P301L)4510 brain. Immunohistochemical studies revealed the progression of abnormal tau conformation and phosphorylation. A, B, Conformational alterations in tau detected with MC-1 are shown in 2.5-month-old rTg(tau_P301L)4510 hippocampus. C, D, At 2.5 months, CP-13-positive labeling was prominent in axons and cell bodies of CA1 hippocampal neurons. Increased numbers of neurons positive for phosphorylation sites associated with the formation of pre-tangles were observed in the hippocampus (E, F, I, J) and cortex (G, H, K, L) of older mice. Original magnifications: A, C, E, G, I, K, 40×; B, D, F, H, J, L, 100×. No positive labeling was observed after parallel processing of control tissue (see supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

Figure 3.

Progression of NFT pathology in rTg(tau_P301L)4510 brain. Formation of Bielschowsky silver-positive NFTs in the rTg(tau_P301L)4510 cortex (Ctx) and hippocampus (Hpp) was age dependent and consistent with forebrain atrophy. A-D, The number of cortical NFTs increased with age, and cortical atrophy was obvious in mice >5 months of age. The arrows in A indicate rare NFTs in the cortex of 2.5-month-old rTg(tau_P301L)4510 mice. Low-power photomicrographs reveal severe hippocampal degeneration with increasing age (E-H), an effect that was most striking in CA1 (I-L). Original magnifications: A-D, 20×; E-H, 4×; I-L, 100×.

Figure 4.

Positive tau labeling in human FTDP brain tissue mirrors rTg(tau_P301L)4510. Parallel immunohistochemical processing of human FTDP-17 (P301L) tissue showed similar staining patterns in the rTg(tau_P301L)4510 brain shown in Figure 2 A-F. G-J, In addition to Bielschowsky staining, standard histological stains Gallyas silver and thioflavin-S confirmed the presence of mature tangles in the 10-month-old rTg(tau_P301L)4510 cortex. Original magnifications: A-H, 100×; I, J, 20×. neg, Negative; pos, positive.

Figure 5.

Age-dependent forebrain degeneration in rTg(tau_P301L)4510 mice. A, Compared with nontransgenic, age-matched control littermates, rTg(tau_P301L)4510 mice exhibited a significant decrease in whole-brain weight from an early age. Because of the small numbers examined at several ages, mice were binned into P1-P14, P28 to 4 months, and 5.5-16 months for statistical analysis. ANOVA revealed that rTg (tau_P301L)4510 mice exhibited a significant decrease in brain weight starting at ∼ 1 month. Data for transgene ANOVA are as follows: P28 to 4 months: F_(1,80) = 37.47, p < 0.0001; 5.5-16 months: F_(1,45) = 130.7, p < 0.0001. Data for tau-negative and tau-positive mice, respectively, are as follows: P1, n = 8, n = 2; P7, n = 8, n = 2; P14, n = 12, n = 4; P28, n = 6, n = 4; 1.3 months, n = 35, n = 9; 2.5 months, n = 17, n = 7; 4 months, n = 3, n = 1; 5.5 months, n = 9, n = 7; 7 months, n = 2, n = 3; 10 months, n = 13, n = 9; 16 months, n = 1, n = 3. Tau-negative mice, 7 months of age, shown here were part of a concurrent study and received doxycycline (200 mg/kg) in their chow starting at 5.5 months of age. Previous studies have shown no effect of doxycycline supplementation on brain weight in nontransgenic mice (SantaCruz et al., 2005). Error bars represent SEM. ^**p < 0.01. B, Macroscopic photographs show forebrain atrophy in a representative 10-month-old tau-Tg mouse compared with control. Histological staining with hematoxylin and eosin (H&E) revealed massive neuronal loss in the frontal cortex and hippocampus in 10-month-old rTg(tau_P301L)4510 mice (D, F) compared with control (C, E). Identical observations were made after immunohistochemical labeling with neuron-specific marker NeuN [tau negative (G, I), tau positive (H, J)]. Neuronal loss in 10-month-old rTg(tau_P301L)4510 mice was accompanied by GFAP-positive reactive astrocytes (L, N), which were rarely observed in age-matched controls (K, M). Positive staining in immersion-fixed control tissue was mainly vascular. Original magnifications: C, D, G, H, 4×; K, L, 20×; E, F, I, J, M, N, 100×. neg, Negative; pos, positive.

Figure 6.

NFT pathology is accompanied by parallel changes in tau biochemistry. Representative immunoblots show soluble and Sarkosyl-insoluble tau species detected in forebrain homogenates of rTg(tau_P301L)4510 mice aged 2.5-10 months. Western blots revealed an age-dependent transition from soluble 55 kDa tau to Sarkosyl-insoluble hyperphosphorylated 64 kDa tau. Human-specific tau species were detected with E1, and equal loading was ensured after probing for GADPH. Positive tau bands were referenced to 64 kDa tau-positive control (P3) and 60 kDa molecular weight markers (arrow).

Figure 7.

Ultrastructure analysis reveals the presence of straight filaments. Representative photomicrographs by electron microscopy on glutaldehyde-fixed brain tissue from a 10-month-old rTg(tau_P301L)4510 mouse are shown. Tau pathology consisted of straight tau filaments that occasionally formed a herring bone pattern. Scale bars: A, 1 μm; B, 500 nm.

Figure 8.

rTg(tau_P301L)4510 mice develop age-dependent memory deficits. Longitudinal studies of rTg(tau_P301L)4510 mice [2.5-9.5 months; n = 26 control and n = 9 rTg(tau_P301L)4510] in the Morris water maze revealed cognitive impairments in retention of spatial reference memory. An independent study examined behavioral performance in 1.3-month-old mice [n = 31 control and n = 10 rTg(tau_P301L)4510]. A, Compared with control mice, the mean time spent swimming in the target quadrant during probe trials decreased in rTg(tau_P301L)4510 mice with increasing age. RMANOVA data are as follows: transgene: F_(1,33) = 80.1, p < 0.0001; age versus transgene: F_(3,99) = 7.46, p < 0.001; age, rTg(tau_P301L)4510 mice: F_(3,24) = 5.82, p < 0.01. Individual quadrant occupancy shows search bias as the percentage of time spent in each of the four quadrants (mean off our probe trials). B, Differences between the percentage of time spent in the target and opposite quadrants compared with all other quadrants was determined using an ANOVA with Fisher's PLSD post hoc analysis. Parallel analyses indicate increasing impairment in spatial navigation in hidden (C) but not cued (D) platform phases of the water-maze protocol. C, RMANOVA data for hidden platform mean path length are as follows: transgene: F_(1,33) = 96.17, p < 0.0001; age versus transgene: F_(3,99) = 12.02, p < 0.0001; age, rTg(tau_P301L)4510 mice: F_(3,24) = 4.64, p = 0.01. During the first 2 d of cued training in the longitudinal study, both nontransgenic and rTg(tau_P301L)4510 mice developed shorter path lengths with repeated testing (age versus transgene RMANOVA: F_(3,99) = 2.58, p = 0.06). D, Although nontransgenic mice also improved with repeated testing during the final day of cued training (age RMANOVA: F_(3,75) = 26.47, p < 0.0001), rTg(tau_P301L)4510 mice exhibited stable performance (age RMANOVA: F_(3,24) = 2.08, p = 0.13). Transgenic control mice aged 7 and 9.5 months shown here were part of a concurrent study and received doxycycline (200 mg/kg) in their chow starting at 5.5 months of age. Previous studies have shown no effect of doxycycline supplementation on spatial reference memory performance in nontransgenic mice (SantaCruz et al., 2005). ^#p < 0.05, ^⋄p < 0.01, ^*p < 0.001, ^**p < 0.0001. neg, Negative; pos, positive; Opp, opposite.

Figure 9.

Loss of motor input induces degeneration of dorsal corticospinal tracts. The spinal cord of 10-month-old mice showed no evident loss of motor neurons in the anterior horn of rTg(tau_P301L)4510 mice (B, D) versus control mice (A, C). However, examination of corticospinal tract tissue indicated a marked loss of neurofilament (E, F). We observed no differences in skeletal muscle integrity when hematoxylin and eosin-processed rTg(tau_P301L)4510 tissue was compared with control (G, H). Most severely affected 10-month-old rTg(tau_P301L)4510 mice displayed a phenotypic grasping reflex in response to tail hang (I, J). Original magnifications: A, B, 4×; C-F, 40×; G, H, 100×. neg, Negative; pos, positive.

Figure 10.

Assessment of motor function in rTg(tau_P301L)4510 mice. Direct assessment of motor ability was measured by analysis of swim speed during each phase of behavioral testing. A-C, Mean swim speeds for mice aged 1.3 months and for mice tested longitudinally from 2.5 to 9.5 months during the visible, hidden, and probe trials, respectively. Age versus transgene RMANOVA data are as follows: A, F_(3,99) = 31.33, p <0.0001; B, F_(3,99) = 14.03, p <0.0001; C, F_(3,99) = 16.30, p <0.0001. Older rTg(tau_P301L)4510 mice at 9. 5 months of age didnot achieve the mean swim speed recorded in tau-negative controls until the final 10 s of probe trials (D, E; transgene RMANOVA, 9.5 months: F_(1,33) = 19.74, p < 0.0001), an effect that was associated with a longer latency to begin swimming (F; age versus transgene RMANOVA: F_(3,99) = 47.55, p < 0.0001; tau-positive age RMANOVA: F_(3,24) = 18.87, p < 0.0001); age versus transgene RMANOVA: F_(3,99) = 47.55, p < 0.0001). Data for 1.3-month-old mice [n = 31 control and n = 10 rTg(tau_P301L)4510] and 2.5- to 9.5-month-old mice [n = 26 control and n = 9 rTg(tau_P301L)4510] are shown. ^#p < 0.05, ^⋄p < 0.01, ^*p < 0.001, ^**p < 0.0001. neg, Negative; pos, positive.