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Attenuated Neurodegenerative Disease Phenotype in Tau Transgenic Mouse Lacking Neurofilaments

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Attenuated Neurodegenerative Disease Phenotype in Tau Transgenic Mouse Lacking Neurofilaments The Journal of Neuroscience, August 15, 2001, 21(16):6026–6035 Attenuated Neurodegenerative Disease Phenotype in Tau Transgenic Mouse Lacking Neurofilaments Takeshi Ishihara, Makoto Higuchi, Bin Zhang, Yasumasa Yoshiyama, Ming Hong, John Q. Trojanowski, and Virginia M.-Y. Lee Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Previous studies have shown that transgenic (Tg) mice overex- particular NFL, resulted in a dramatic decrease in the total pressing human tau protein develop filamentous tau aggre- number of tau-positive spheroids in spinal cord and brainstem. gates in the CNS. The most abundant tau aggregates are found Concomitant with the reduction in spheroid number, the bigenic in spinal cord and brainstem in which they colocalize with mice showed delayed accumulation of insoluble tau protein in neurofilaments (NFs) as spheroids in axons. To elucidate the the CNS, increased viability, reduced weight loss, and improved role of NF subunit proteins in tau aggregate formation and to behavioral phenotype when compared with the single T44 Tg test the hypothesis that NFs are pathological chaperones in the mice. These results imply that NFs are pathological chaperones formation of intraneuronal tau inclusions, we crossbred previ- in the development of tau spheroids and suggest a role for NFs ously described tau (T44) Tg mice overexpressing the smallest in the pathogenesis of neurofibrillary tau lesions in neurodegen- human tau isoform with knock-out mice devoid of NFL erative disorders that contain both NFs and tau proteins. (NFLϪ/Ϫ) or NFH (NFHϪ/Ϫ). Depletion of NF subunit proteins Key words: tau; neurofilaments; neurodegeneration; neurofi- from the T44 mice (i.e., T44;NFLϪ/Ϫ and T44;NFHϪ/Ϫ), in brillary pathology; animal models; cytoskeleton Filamentous tau inclusions, accompanied by extensive neuron esis of tauopathies, we generated transgenic (Tg) mice that over- loss and gliosis, are the neuropathological hallmarks of an ex- expressed the shortest human brain tau isoform (T44) in CNS panding family of neurodegenerative diseases that are now col- (Ishihara et al., 1999, 2001). As reported previously, the T44 Tg lectively known as tauopathies (Lee et al., 2001). Prototypical mice acquired age-dependent CNS pathology similar to the fila- tauopathies are exemplified by frontotemporal dementia with mentous tau inclusions found in FTDP-17 and ALS/PDC, includ- parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic ing insoluble, hyperphosphorylated intraneuronal aggregates lateral sclerosis/parkinsonism–dementia complex (ALS/PDC), formed by tau-immunoreactive filaments (Ishihara et al., 1999). Alzheimer’s disease (AD), and progressive supranuclear palsy These inclusions, mostly in the form of spheroids, were abundant (PSP). Like other tauopathies, FTDP-17, PSP, AD, and ALS/ in spinal cord neurons, in which they were associated with axon PDC are characterized by numerous inclusions formed by aggre- degeneration and reduced axonal transport in ventral roots, as gated paired helical filaments (PHFs) and/or straight filaments well as spinal cord gliosis and motor weakness. Thus, these Tg composed of aberrantly phosphorylated tau proteins (PHF-tau) mice recapitulate some of, but not all, key aspects of prototypical in widespread regions of the CNS (Hong et al., 2000). The tauopathies. For example, tau-positive spheroids are strongly discovery of tau gene mutations in FTDP-17 kindreds provides positive for neurofilaments (NFs) in the spinal cord and brain- unique opportunities to elucidate the disease mechanisms that stem of T44 Tg mice. Significantly, NF subunit proteins have long underlie FTDP-17 as well as related brain disorders character- been associated with pathogenic protein aggregates in a number ized by abundant insoluble intracellular filamentous tau inclu- of neurodegeneration diseases. For example, spheroids in the T44 sions, including AD (Clark et al., 1998; Hong et al., 1998; Hutton Tg mice and in the spinal cord of ALS/PDC patients contain both et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998). The tau and NF-immunoreactive filaments, and all three NF subunits FTDP-17 mutations occur in exons and introns of the tau gene, (NFL, NFM, and NFH) are found in neurofibrillary tangles and they may cause FTDP-17 by altering the functions or levels (NFTs) at late stages of the disease in AD patients (Schmidt et of specific tau isoforms in the CNS (Hong et al., 1998; Hutton et al., 1989; Ishihara et al., 1999). NFs are also detected in Lewy al., 1998; D’Souza et al., 1999). bodies in which they coexist with ␣-synuclein (Tu et al., 1998). To begin elucidating the role of tau inclusions in the pathogen- Based on these and other observations, we hypothesize that NFs may play a role as “pathological chaperones” (Wisniewski and Received March 21, 2001; revised May 11, 2001; accepted May 30, 2001. Frangione, 1992) in facilitating the aggregation of tau in our Tg This work was supported by grants from the National Institute on Aging. We mice and in the human neurodegenerative diseases discussed thank Drs. Jean-Pierre Julien and Robert Lazzarini for providing the NFLϪ/Ϫ and above. NFHϪ/Ϫ mice, respectively, and the Biomedical Imaging Core Facility of the University of Pennsylvania for assistance in the electron microscopic studies. To test this hypothesis, we generated bigenic mice overexpress- Correspondence should be addressed to Dr. Virginia M.-Y. Lee, Department of ing tau protein but lacking NFL (Zhu et al., 1997) or NFH (Elder Pathology and Laboratory Medicine, University of Pennsylvania School of Medi- et al., 1998) by crossbreeding T44 Tg mice with NFL knock-out cine, Maloney 3, HUP, 3600 Spruce Street, Philadelphia, PA 19104-4283. E-mail: [email protected]. (NFLϪ/Ϫ) or NFH knock-out (NFHϪ/Ϫ) mice, respectively. Copyright © 2001 Society for Neuroscience 0270-6474/01/216026-10$15.00/0 Our studies showed that depletion of NF subunit proteins, espe- Ishihara et al. • Tau Transgenic Mouse Lacking Neurofilaments J. Neurosci., August 15, 2001, 21(16):6026–6035 6027 Research. Immunohistochemistry were performed on representative 6 ␮m paraffin sections from brain, spinal cord, and spinal roots. Three animals from each mouse line and at each specific age were used for the immunohistochemical studies. Anti-NF antibodies were used to detect the presence or absence of NF subunit proteins in the CNS of Tg mice, and sections were also stained with anti-tau antibodies to localize the tau aggregates (Table 1). In addition, mouse monoclonal antibodies (mAbs) to ␣- and ␤-tubulin and antibodies to ␣-internexin and to peripherin were used to assess whether or not these molecules also accumulate in tau aggregates (Table 1). Axonal tau pathology in Tg mice was quantified by counting the number of tau-positive spheroids with a diameter of Ͼ5 ␮m. This quantitative analysis was performed for 20 lumber spinal cord sections from each Tg mouse, and the average number of spheroids per section was used as a representative value. The mean and SD values of the number of spheroids in three Tg mice at the same age were estimated in each line, and the difference in the number of spheroids among all of the lines was examined statistically by one-way ANOVA. To see colocalization of the cytoskeletal components in the spheroids, selected sections were also double- and triple-labeled by immunofluores- cence using anti-tau, anti-NF subunit, and anti-tubulin antibodies (Table 1). Western blot analysis of Tau, NFs, peripherin, ␣-internexin, and tubulin expressed in the CNS of Tg mice. Tissues were carefully dissected after mice were lethally anesthetized. The tissues were homogenized in 2 ml/gm ice-cold high salt reassembly buffer (RAB) [0.1 M MES,1mM EGTA, 0.5 mM MgSO4, 0.75 M NaCl, 0.02 M NaF, 1 mM PMSF, and 0.1% Figure 1. Analysis of protein expression in cortices. Western blot anal- protease inhibitor cocktail (100 ␮g/ml each of pepstatin A, leupeptin, ysis of tau, NFs, and ␣-tubulin isolated from cortices of the different lines N-tosyl-L-phenylalanyl cloromethyl ketone, N-tosyl-lisine cloromethyl ke- of mice. Six-month-old mice from each group were used. 17026, an tone, soybean trypsin inhibitor, and 100 mM EDTA), pH 7.0] and cen- anti-recombinant tau antibody that recognizes both human and mouse trifuged at 50,000 ϫ g for 40 min at 4°C in the Beckman Instruments tau, shows the expression level of tau in each line of mice. Endogenous (Fullerton, CA) TL-100 ultracentrifuge. The pellets were saved for mouse tau protein levels are comparable in WT, NFLϪ/Ϫ, and NFHϪ/Ϫ NF–peripherin–internexin analyses. Half of the supernatants were saved mice, and overexpressed human tau protein levels in T44 Tg, T44 Tg; for tubulin analyses, and the rest were boiled for 5 min, chilled on ice for NFLϩ/Ϫ, T44;NFLϪ/Ϫ, T44;NFHϩ/Ϫ, and T44;NFHϪ/Ϫ mice were 5 min, and recentrifuged at 10,000 ϫ g for 20 min at 4°C, and the ϳ10-fold higher than endogenous mouse tau. NFH and NFM protein supernatant were saved for tau Western blotting. Protein concentration levels were decreased ϳ85 and 50%, respectively, in the cortices of was then determined for the samples using the BCA assay kit (Pierce, NFLϪ/Ϫ and T44 Tg;NFLϪ/Ϫ mice, whereas the level of ␣-tubulin was Rockford, IL). Equal amounts of samples were subsequently resolved on dramatically increased compared with WT mice. NFL protein levels were 7.5% SDS-PAGE gels and transferred onto nitrocellulose membranes. decreased ϳ30% in NFHϪ/Ϫ and T44 Tg;NFHϪ/Ϫ mice without a Quantitative Western blot analyses (three animals from each line) were change in NFM protein levels. Equal amounts (10 ␮g for tau; 20 ␮g for performed by using either [ 125I]-labeled goat anti-mouse IgG or [ 125I]- NFs and ␣-tubulin) of mouse cortical samples were loaded on each gel labeled Protein A (NEN, Boston, MA) as secondary antibodies as lane.
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