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Comparative Study
. 2008 Apr;27(7):1615-25.
doi: 10.1111/j.1460-9568.2008.06161.x.

Tau expression levels from various adeno-associated virus vector serotypes produce graded neurodegenerative disease states

Ronald L Klein  1 , Robert D Dayton, Jason B Tatom, Cynthia G Diaczynsky, Michael F Salvatore
Affiliations
Comparative Study

Tau expression levels from various adeno-associated virus vector serotypes produce graded neurodegenerative disease states

Ronald L Klein et al. Eur J Neurosci. 2008 Apr.

Abstract

Neurodegenerative diseases involving neurofibrillary tangle pathology are pernicious. By expressing the microtubule-associated protein tau, a major component of tangles, with a viral vector, we induce neuropathological sequelae in rats that are similar to those seen in human tauopathies. We tested several variants of the adeno-associated virus (AAV) vector for tau expression in the nigrostriatal system in order to develop models with graded onset and completeness. Whereas previous studies with AAV2 tau vectors produced partial lesions of the nigrostriatal system, AAV9 or AAV10 tau vectors were more robust. These vectors had formidable efficacy relative to 6-hydroxydopamine for dopamine loss in the striatum. Time-courses for tau transgene expression, dopamine loss and rotational behavior tracked the disease progression with the AAV9 tau vector. There was a nearly complete lesion over a delayed time-course relative to 6-hydroxydopamine, with a sequence of tau expression by 1 week, dopamine loss by 2 weeks and then behavior effect by 3-4 weeks. Relative to AAV2 or AAV8, tau expression from AAV9 or AAV10 peaked earlier and caused more dopamine loss. Varying vector efficiencies produced graded states of disease up to nearly complete. The disease models stemming from the AAV variants AAV9 or AAV10 may be useful for rapid drug screening, particularly for tau diseases that affect the nigrostriatal system, such as progressive supranuclear palsy.

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Figures

FIG. 1
FIG. 1
Comparative expression from AAV2, AAV8, AAV9 and AAV10 tau vectors at (A) 1 week, (B) 2 weeks and (C) 4 weeks. (D) Quantification of human tau expression, normalized to a constitutive gene product [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] expression (N) (*significant serotype differences, P < 0.05, anovas/Bonferroni’s multiple comparison tests, see Results for all comparisons). The dissected SN, the injected area, showed relatively strong and rapid expression from AAV9 or AAV10. AAV2 was weaker than either AAV9 or AAV10 at all intervals (P < 0.05). AAV8 expression was similar to AAV2 at 1 and 2 weeks but was greater than AAV2 by 4 weeks (P < 0.001), by which time AAV8 expression was similar to AAV9 or AAV10. Each sample is shown; equal proteins were loaded on gels. AAV2 or AAV8 was run at a vector dose of 6.4 × 109 vg, whereas AAV9 or AAV10 was run at a lower dose of 3 × 109 vg.
Fig. 2
Fig. 2
Neurochemical data from rat striatum. (A) DA levels on the treated side relative to the untreated side of the brain, expressed as a ratio, were completely depleted by 6-OHDA at 1 and 4 weeks post-treatment. By 4 weeks, AAV8 tau reduced DA levels to a greater extent than AAV2 tau (P < 0.001, anova/Bonferroni’s multiple comparison test). AAV9 tau or AAV10 tau reduced DA more rapidly as each was different from AAV2 tau at 2 weeks (P < 0.001 for each) and both AAV9 and AAV10 tau produced greater loss than AAV8 tau at both 2 and 4 weeks (P < 0.001–0.05 for different comparisons). N = 3–11/data point. (B) The 4 week data from A, shown with control AAV8 or AAV9 GFP vectors. N-value indicated, as for C. (C) 6-OHDA drove up DA turnover as expected, as did AAV9 tau at 4 weeks (P < 0.001 compared with AAV9 GFP). *Difference between AAV tau and its cognate GFP vector (P < 0.001–0.01).
Fig. 3
Fig. 3
AAV9 GFP expression in the nigrostriatal pathway. (A) GFP fluorescence at 4 weeks after injection of AAV9 GFP to the SN at a dose of 3 × 109 vg. (B) The same section as A counterstained for TH showing good overlap with the GFP in A and the pars compacta DA neurons. (C) GFP was expressed unilaterally in corpus striatum after unilateral injections as in A. Scale bars: A and B, 132 µm; C, 528 µm.
Fig. 4
Fig. 4
Transgene expression and effects on DA neurons. Examples of transgene expression at 4 weeks after injections of (A) AAV9 GFP, (B) AAV9 tau, (C) AAV10 tau and (D) AAV9 alpha-synuclein. All of the vectors were injected at a dose of 3 × 109 vg. GFP fluorescence in the SN (A) or human alpha-synuclein immunoreactivity (D) was dense in the SN pars compacta, whereas with the tau vectors in B and C there was evidence of human tau staining in the pars reticulata but little or no remaining expression in the pars compacta. TH staining after unilateral injections of (E) AAV9 GFP, (F) AAV9 tau, (G) AAV10 tau and (H) AAV9 alpha-synuclein. The right side of the panel is the injected side for E–H. At this vector dose, TH was preserved in the GFP and alpha-synuclein groups in E and H, along with the transgene expression in the pars compacta above, whereas AAV9 or AAV10 tau vectors in F and G obliterated TH on the injected side. Scale bars: A–D, 90 µm; E–H, 360 µm.
Fig. 5
Fig. 5
Stereology estimates of SN DA neurons. AAV9 GFP vector did not alter numbers of DA neurons at 4 weeks relative to uninjected SN but AAV9 tau or AAV10 tau reduced DA neuron profiles by 88–94%. *P < 0.001, anova/Bonferroni’s multiple comparison test, N = 5–6/ group.
Fig. 6
Fig. 6
AAV9 tau gene transfer obliterates GFP, TH and neuronal nuclei (NeuN) markers, demonstrating loss of DA neurons rather than solely loss of TH. A mixture of AAV9 GFP/lactated ringer’s solution on one side of the brain (A–C) or AAV9 GFP/AAV9 tau (D–F) was injected 4 weeks earlier. Without tau, there was GFP (A) and TH (B) expression and NeuN (C) present in the SN pars compacta on adjacent sections. With tau expression, there were fewer GFP- (D) and TH- (E) expressing neurons, and fewer neuronal nuclei (F) in the SN pars compacta on adjacent sections. Camera exposure times were equal for each side of the brain. Scale bar, 150 µm.
Fig. 7
Fig. 7
TH fiber density in striatum. A series of five sections from one rat is shown for each vector group. The left hemisphere is the injected side, showing marked loss of striatal TH fiber density in the 6-OHDA, AAV8 tau, AAV9 tau or AAV10 tau vector groups.
Fig. 8
Fig. 8
Quantification of TH fiber density in striatum. The TH staining in striatum ipsilateral to the injection in the SN was measured as a ratio to the staining on the uninjected contralateral side. AAV8 (P < 0.01) or AAV9 (P < 0.001) tau caused loss of striatal TH immunoreactivity relative to its matching GFP vector at 4 weeks (*anova/Bonferroni’s multiple comparison test). The loss was greater with either AAV9 tau (P < 0.01) or AAV10 tau (P < 0.05) than AAV2 tau (N = 3–4/group).
Fig. 9
Fig. 9
Amphetamine-stimulated rotational behavior. (A) Rotational behavior at 1 week prior to and 2, 3 and 4 weeks after AAV9 GFP gene transfer (N = 3). (B) Rotational behavior before and after AAV9 tau gene transfer (N = 6). There was a signficiant interaction in the repeated-measures anova for turning direction over time in the AAV9 tau group (P < 0.0001). (C) 4 week data, N-value indicated. There was a significant turning bias in the AAV9 tau group (*P < 0.0001, t-test).
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