Axonal degeneration induced by targeted expression of mutant human tau in oligodendrocytes of transgenic mice that model glial tauopathies

Abstract

Abundant filamentous tau inclusions in oligodendrocytes (OLGs) are hallmarks of neurodegenerative tauopathies, including sporadic corticobasal degeneration and hereditary frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). However, mechanisms of neurodegeneration in these tauopathies are unclear in part because of the lack of animal models for experimental analysis. We address this by generating transgenic (Tg) mice expressing human tau exclusively in OLGs using the 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter. Filamentous OLG tau inclusions developed in these Tg mice as a result of human tau expression in OLGs, especially those expressing the FTDP-17 human P301L mutant tau. Notably, structural disruption of myelin and axons preceded the emergence of thioflavin-S positive tau inclusions in OLGs, but impairments in axonal transport occurred even earlier, whereas motor deficits developed subsequently, especially in Tg mice with the highest tau expression levels. These data suggest that the accumulation of tau in OLG cause neurodegeneration, and we infer they do so by disrupting axonal transport. We suggest that similar defects may also occur in sporadic and hereditary human tauopathies with OLG tau pathologies.

Figure 1.

Expression of Tg human tau in OLGs of the murine nervous system. A, Schematic representation of the transgene construct used to generate tau Tg mice, which consists of a stop codon, human T34 with or without the PL mutation, and polyadenylation site (Poly-[A]) inserted into HindIII site between CNP exons 0 and 1 (E0 and E1). Transgene expression in OLGs was directed by the CNP promoter P1, whereas expression from CNP promoter P2 was impeded by a stop codon upstream of the T34 sequence. B, Immunoblots using the human tau-specific anti-tau antibody T14 demonstrate varying levels of transgene expression in the brains of the different tau Tg lines. All protein samples were derived from 3-month-old heterozygous Tg mice. C, D, Sections of the basal ganglia (C) and spinal cord (D) of a 3-month-old line 12 PL Tg mouse immunolabeled with antibody 17026 show intense tau immunoreactivity in glial cells with the morphology of OLGs, whereas neurons show far less intense positivity for endogenous mouse tau (arrow). E-G, Double-immunofluorescence staining with anti-CNP antibody (E) and anti-tau antibody 17026 (F) in spinal cord of a 3-month-old line 12 PL Tg mouse showing robust expression of tau in CNP-positive OLGs. A dualchannel image (G) indicates ∼100% colocalization of CNP and tau positivity in OLGs. Scale bars: C, D, 100 μm; insets in C, D, 20 μm; E-G, 50 μm.

Figure 2.

Formation of fibrillary human tau inclusion in OLGs of PL Tg mice as a function of age. A, T14 immunoblots of human tau in HS-, RIPA-, and FA-soluble fractions extracted from the brains (top row) and spinal cords (bottom row) of line 39 hWT and line 12 PL Tg mice at different ages. Levels of HS-soluble tau remained stable during aging, as was the case for RIPA- and FA-extractable tau proteins in the hWT Tg mice, but there was a pronounced increase in these relatively insoluble species of tau in older PL Tg mice. The retarded electrophoretic mobility of the FA-extractable tau in older PL Tg mice is consistent with its increased phosphorylation. B-D, Fibrillary lesions in presumptive OLGs in a Gallyas silver-stained spinal cord section of 15-month-old line 12 PL Tg mouse. High-power photomicrographs of the inclusions in the anterior horn (C) and anterior column (D) reveal that pathological fibrillar tau extends into proximal processes of OLGs. E, Similar OLG inclusion labeled with thioflavin-S [counterstained for nuclei with 4′,6′-diamidino-2-phenylindole (DAPI)] in 15-month-old line 12 PL Tg mouse. F, Gallyas silver-positive coiled bodies in the frontal white matter of a patient with CBD, showing resemblance of fibrillary lesions in the PL Tg mice to those in FTD patients. Scale bars: B, 50 μm; C-F, 20 μm.

Figure 3.

Progressive degeneration of OLGs and axons in the spinal cord of PL Tg mice. A-H, Age-associated loss of myelin in the posterior column of the spinal cord revealed by Luxol fast blue staining on 6- (A, E), 9- (B, F), and 12- (C, G) month-old PL Tg and 12-month-old WT non-Tg mice (D, H). High-power view photomicrographs are displayed in the bottom panels (E-H). I-L, Disrupted myelin accompanies axonal damage in the posterior column of the spinal cord. Double-immunofluorescence staining of a frozen section from 6-month-old PL Tg mouse using anti-CNP (I) and anti-NFH (RMO24) (J) antibodies demonstrates disorganization of myelin and atrophy or loss of axons as indicated by arrows on a merged image (K; counterstained with DAPI), whereas a WT non-Tg mouse (L) at the same age does not show these pathological alterations. M-R, Impaired kinesin transport in spinal cord OLGs of PL Tg mouse at 9 months of age. Photomicrographs of doubly stained OLGs of an age-matched WT mouse with channels for kinesin (M), CNP (N), and merged images (O; counterstained with DAPI) depict normal physiological distribution of kinesin that ranges from the cell body to distal processes (arrow in M) in a CNP-positive OLG. Arrowhead in M indicates kinesin in the axon. Unlike WT mice, PL Tg mouse showed reduced kinesin staining in distal processes of OLGs (arrow in P), which were filled with tau immunoreactivity (Q). Multichannel photomicrograph demonstrates disrupted kinesin staining in tau-positive OLGs (R; counterstained with DAPI). S, Loss of myelin components in the brains of 12-month-old PL Tg mice visualized by Coomassie blue. Myelin samples extracted from line 2 and line 12 mice contained significantly less myelin constituents, including MBP (arrowheads) and CNP (arrows), than age-matched WT mouse-derived sample. Other minor bands are also less intense in the PL Tg mice than those in the WT mouse, implying uniform reduction of myelin proteins as a result of OLG tau pathology. Scale bars: A-D, 200 μm; E-H, 100 μm; I-L, P-R, 20 μm.

Figure 4.

Ultrastructural alterations of myelin and axons associated with accumulation of abnormal tau filaments in the spinal cords of the line 12 PL Tg mice at 12 months of age. A-C, Myelin pathology and axonal degeneration demonstrated by TEM. Although the WT mouse did not develop changes in myelin and axons (A), myelin in the PL mouse is fragmented and disorganized (arrows in B), and the axons within the myelin sheaths are severely atrophic. Disruption of myelin and axon loss are shown at higher magnification (C). D-F, Accumulation of tau-immunoreactive filaments in the PL Tg mouse revealed by immuno-EM with anti-tau antibody 17026. Abnormal filaments were present between detached myelin layers (D) and in areas apposed to degenerating myelin lacking a central axon (arrow in D). High-power view shows intense labeling of well oriented filaments with anti-tau antibody (E). Tau-immunolabeled filaments were also localized to OLG processes (arrow in F), whereas filaments in axons were not tau positive and had side arms characteristic of NFs (arrowhead in F). Scale bars: A, B, 2 μm; C, D, 1 μm; E, F, 500 nm.

Figure 5.

Biochemical impact of OLG tau accumulations on axons in the optic nerve of the PL Tg mice. A, Overexpression of Tg tau in the optic nerve interfascicular OLGs shown by double-immunofluorescence staining with anti-CNP antibody (A, C) and anti-tau antibody 17026 (B, D) for 6-month-old WT non-Tg (A, B) and line 12 PL tau Tg (C, D) mice. In contrast to diffuse tau immunoreactivity in WT OLGs, strong tau staining is seen in specific CNP-positive OLGs of the PL Tg mouse. CNP staining in myelin and tau staining in axons were moderately diminished in the PL Tg mouse compared with the WT mouse. E-G, Immunoblotting for optic nerve samples with antibodies against phosphorylated NFH (NFH p++), NFM, NFL, α-tubulin (α-tub) (E), and endogenous mouse tau (F), and quantification of the blots (n = 4 in each group) (G). Substantial reduction of NFH and NFM occurred in the line 2 and line 12 PL Tg mice at 4 months of age (E and top and middle panels in G). The 12-month-old PL Tg mice also showed slight decrease in the amounts of NFL and α-tubulin. There were no remarkable differences in the levels of low-molecular-weight tau isoforms ranging from 50 to 70 kDa (indicated by arrows) between the WT and PL Tg mice, whereas middle-molecular-weight tau (∼100 kDa, indicated by an asterisk) in the PL Tg mice was pronouncedly reduced relative to the WT mice (F and bottom panel in G). ^*p < 0.05 by multiple comparison by ANOVA. Scale bar: A-D, 50 μm.

Figure 6.

Impaired axonal transport in the optic nerves of the PL Tg mice before ultrastructural disruption of myelin and axons. A-F, TEM of the optic nerve sections of line 12 PL mice at 6 (A) and 9 (B, C) months of age, WT mice at 6 (D) and 9 (E) months, and line 39 h WT tau Tg mouse at 9 months (F). The 6-month-old PL tau Tg mouse exhibited relatively normal myelin sheaths and axons morphologically (A), whereas there were marked losses of these structures and expansion of interstitial space in the 9-month-old PL Tg mice (B). Severe disorganization of myelin and axonal atrophy are also shown in a high-power view (C). The WT and hWT Tg mice did not develop overt pathologies until 9 months of age (D-F). G-L, Quantitative assays of fast (G, I, J) and slow (H, K, L) axonal transport in the optic nerve. SDS-PAGE of protein samples from optic nerve segments indicates significant retardation of fast axonal transport in 6-month-old line 12 PL Tg mice compared with age-matched WT mice (G, I). This deficit became greater at 9 months of age (J). The arrow in G identifies a major protein band undergoing fast axonal transport and is used here as a marker for the quantification of fast transport. Slow axonal transport of proteins such as tubulin and NFL (corresponding bands are indicated in H) were also significantly slower in the 6-month-old PL Tg mice relative to the WT mice (K, L). Error bars in I-L represent SE (n = 3 in each group). ^*p < 0.05 by t test. Scale bars: A, B, D-F, 2 μm; C, 1 μm.

Figure 7.

Weight loss and progressive motor deficits in the PL Tg mice. A, Weights of different lines of PL and hWT Tg mice at 12 months of age. Values are normalized by the mean body weight of the WT mice at the same age. Homozygotes of the line 7 PL Tg mice are not included, because most of them did not survive until this age. n = 6 in each group. B, Frequency of limb twitching in tail suspension test. Although the hWT Tg mice (left) showed this pathologic motor phenotype at a frequency that was higher than that of WT mice reported previously (Higuchi et al., 2002a), the frequency was further augmented in the PL mice (right). The emergence of limb twitching was approximately proportional to the dosage of the transgenes in both of the hWT and PL Tg mice.

Figure 8.

Oligodendrocytic tau accumulation and disruption of myelin sheaths in patients with FTD. A, Frozen section of the frontal white matter from a PSP patient immunolabeled with antibodies against CNP (green) and tau (PHF1, red) and counterstained with DAPI (blue). Myelin sheaths that were connected to a phospho-tau-immunoreactive OLG showed marked disorganization (arrows), although a large portion of myelin appeared intact (arrowhead). B, C, Paraffin sections of the frontal white matter from an FTD patient stained with PHF1 (B) and Luxol fast blue (C). Tau-positive inclusions were frequently found in putative OLGs (B). Round, well bordered deposits were localized to the cytoplasm of OLG as revealed in high-power photomicrograph (inset in B). Severe loss of myelin was also observed in the same region (C). Scale bars: A, inset in B, 15 μm; B, 25 μm; C, 100 μm.