Transcriptome analysis of a tau overexpression model in rats implicates an early pro-inflammatory response

Abstract

Neurofibrillary tangles comprised of the microtubule-associated protein tau are pathological features of Alzheimer's disease and several other neurodegenerative diseases, such as progressive supranuclear palsy. We previously overexpressed tau in the substantia nigra of rats and mimicked some of the neurodegenerative sequelae that occur in humans such as tangle formation, loss of dopamine neurons, and microgliosis. To study molecular changes involved in the tau-induced disease state, we used DNA microarrays at an early stage of the disease process. A range of adeno-associated virus (AAV9) vector doses for tau were injected in groups of rats with a survival interval of 2 weeks. Specific decreases in messages for dopamine-related genes validated the technique with respect to the dopaminergic cell loss observed. Of the mRNAs upregulated, there was a dose-dependent effect on multiple genes involved in immune response such as chemokines, interferon-inducible genes and leukocyte markers, only in the tau vector groups and not in dose-matched controls of either transgene-less empty vector or control green fluorescent protein vector. Histological staining for dopamine neurons and microglia matched the loss of dopaminergic markers and upregulation of immune response mRNAs in the microarray data, respectively. RT-PCR for selected markers confirmed the microarray results, with similar changes found by either technique. The mRNA data correlate well with previous findings, and underscore microgliosis and immune response in the degenerative process following tau overexpression.

Fig 1

AAV capsid protein western blot. Each viral dose used in the study was loaded and probed with an antibody against the AAV viral proteins VP1, VP2, and VP3 (indicated on right side of panel; size markers in kDa on left side), to ensure equal viral particles for the microarray study. Each matching dose of specific vectors had similar bands, and the intensity of the bands matched with the vector genome amounts loaded.

Fig 2

RT-PCR validation of selected mRNAs from the microarray study: tyrosine hydroxylase (TH), dopamine transporter (DAT), RT1-Db (involved in antigen presentation), interleukin 1β (IL 1B), tumor necrosis factor (TNF). The amount of each target mRNA was normalized by subtracting the signal for a housekeeping mRNA (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) from an aliquot of the same sample. Data are shown for the dose matching empty vector and tau groups (1.2 × 10¹⁰ vector genomes) and a higher dose of tau vector (3.6 × 10¹⁰ vector genomes) with 3-4 animals per group. In all cases, there were group differences by ANOVA with dose-dependent differences between the tau groups in Bonferroni post-tests (TH, DAT, P < 0.05; RT1-Db, P < 0.01; IL-1β, TNF, P < 0.001). High dose tau vector caused decreases in TH and DAT and increases in RT1-Db, IL-1B, and TNF, although all of the data are expressed as negative values as a result of the high levels of GAPDH.

Fig 3

Histological analyses two weeks after injections of AAV9 GFP or AAV9 tau to the substantia nigra (SN). Results from the low and mid doses of the microarray study (2.4 × 10⁹ or 1.2 × 10¹⁰ vector genomes) are shown for each vector. (A-C) GFP epifluorescence, TH immunoreactivity, and staining for the microglial marker Cd11b on adjacent sections after low dose AAV9 GFP injection. TH staining was similar to the uninjected contralateral side (not shown), and pronounced microglial staining was restricted to the needle track. Labels for the SN pars compacta and pars reticulata are provided in A. (D-F) There was an apparent dose-dependent increase in both the spread and intensity of GFP expression with the higher dose, although both TH and Cd11b staining were similar at both doses of AAV9 GFP. (G-I) Hyperphosphorylated tau immunoreactivity (CP13 antibody) after AAV9 tau injection was found mainly in the SN pars compacta, and both TH and Cd11b staining appeared similar to results with both doses of the GFP vector. (J-L) In contrast, the higher dose of tau resulted in spread of hyperphosphorylated tau into the pars reticulata, but the pars compacta was partially blank for tau staining, likely due to dopaminergic cell loss in that area, consistent with reduced TH immunoreactivity in the SN. The microglial staining pattern was distinct with the higher dose of tau, with clear microgliosis away from the needle track in the SN. A higher dose of AAV9 tau (3.6 × 10¹⁰ vector genomes) resulted in even more pronounced effects on TH and Cd11b (not shown). Camera settings for the fluorescent images in A, D or C, F, I, L were kept equal. Bar in A = 134 μm.