Experimental evidence for the age dependence of tau protein spread in the brain

Abstract

The incidence of Alzheimer's disease (AD), which is characterized by progressive cognitive decline that correlates with the spread of tau protein aggregation in the cortical mantle, is strongly age-related. It could be that age predisposes the brain for tau misfolding and supports the propagation of tau pathology. We tested this hypothesis using an experimental setup that allowed for exploration of age-related factors of tau spread and regional vulnerability. We virally expressed human tau locally in entorhinal cortex (EC) neurons of young or old mice and monitored the cell-to-cell tau protein spread by immunolabeling. Old animals showed more tau spreading in the hippocampus and adjacent cortical areas and accumulated more misfolded tau in EC neurons. No misfolding, at any age, was observed in the striatum, a brain region mostly unaffected by tangles. Age and brain region dependent tau spreading and misfolding likely contribute to the profound age-related risk for sporadic AD.

Fig. 1. Enhanced neuron-to-neuron propagation of mutant P301Ltau in vivo.

(A) Schematic showing the AAV sequence, the mRNA, and the proteins encoded in AAV CBA.eGFP.2a.huTau (WTtau and P301Ltau), as well as the tau protein propagation principle and detection methodology. Using a self-cleaving 2a peptide, transduced “donor” neurons express both eGFP and human tau as individual proteins. The propagation of tau can be visualized by immunofluorescence labeling of postmortem brain sections or fixed neurons in culture: Human tau detected in “recipient” neurons that do not express the fluorescence transduction marker eGFP indicates the propagation of tau between cells. Thereby, the upstream location of the GFP transduction marker prevents the detection of false positives that could occur due to incomplete translation of the mRNA. (B) Schematic for the unilateral injection of AAV eGFP-2a-huTau into the EC (green) and the experimental work flow. The location of the EC, the HPC (and subregion CA1), and the dentate gyrus (DG) in horizontal mouse brain sections is indicated. (C) Example of the immunofluorescence labeling of human tau (red) and GFP (green) in a horizontal brain section of a GFP-2a-WTtau–injected mouse with many huTau recipient cells: [huTau⁺/GFP⁻] cells, which received tau by cell-to-cell propagation (white arrowheads in close-ups 1 and 2), can be seen adjacent to the EC injection side (1) and in synaptic connected area CA1 (2). Cells transduced with the AAV (GFP⁺) can, in this specific case, be found in the EC and CA3. (D) Quantification of tau propagation (no. of recipient cells/no. of transduced cells) was done by counting all recipient (huTau⁺, red) and donor neurons (GFP⁺, green) in the entire ipsilateral EC and HPC formation (dashed white line). A higher propagation (P = 0.0472) was detected for P301Ltau compared to WTtau. (E) Scatterplot shows the average of recipient versus donor cells per mouse. Mean ± SEM, n = 3 animals per group and n = 4 to 5 brain sections per animal; single data points represent the mean per animal; unpaired two-tailed Student’s t test with Welch’s correction.

Fig. 2. Propagation and misfolding of tau are enhanced with age.

(A) Representative images of human P301Ltau propagation after AAV GFP-2a-P301Ltau injection into the EC in young and old mice. For analysis, huTau recipient neurons (white arrowhead in CA1 close-up) were counted in the EC, the hippocampal formation, and the adjacent cortex (ROI outlined with dashed white line). (B) Tau propagation (no. of recipient cells/no. of transduced cells) in old animals was still small but threefold higher (P = 0.0322) compared to young adult animals (12 weeks after injection). (C) The amount of transduced GFP⁺ neurons in the EC of young and old mice relative to the amount of total neurons (DAPI⁺ neuronal nuclei) was determined by counting nuclei and GFP⁺ cells in the same area of EC in parallel. (D) Number of neurons (large DAPI⁺ nuclei; smaller glia nuclei were neglected) in the injected (ipsi) and noninjected (contra) EC of young and old mice reveals no difference in neuronal numbers. (E) Number of microglia (Iba1⁺) in the HPC of the ipsilateral and contralateral hemisphere shows no difference between young and old mice. (F) GFAP levels in EC/HPC extracts from young and old mice (6 weeks after injection) suggest more activated astrocytes in the ipsilateral side of both young and old mice, compared to the contralateral side. (G) Western blot analysis of EC/HPC extracts from noninjected young and old mice reveals no general difference in proteostasis, in the ER stress marker CHOP, or in autophagy markers LC3B and p62. (H) Images showing transduced neurons (GFP⁺, green) in the EC having misfolded tau (Alz-50⁺, pink) in an old and a young animal. (I) The amount of Alz-50⁺ neurons in the EC (normalized to area covered by transduced GFP⁺ neurons) appears to be ~2-fold higher in old animals (ns, not significant; P = 0.1531). Data presented as mean ± SEM. For cell counts, n = 4 young and 5 old animals and n = 3 to 5 brain sections per animal; single data points represent the mean per animal. For brain lysates, n = 5 young and old animals. Two-tailed Student’s t test with Welch’s correction when comparing two groups, and one-way ANOVA with Sidak’s correction when comparing multiple groups.

Fig. 3. P301Ltau misfolding in EC but not striatal neurons.

(A) Injection schematic and representative immunofluorescently labeled horizontal brain section of mice that were injected in both the EC and the striatum (CPu). Reference images of brain sections (coronal and horizontal) were taken from the MBL mouse atlas (www.mbl.org/atlas232/atlas232_frame.html). (B) Brain sections and close-ups of P301Ltau-expressing neurons in the injection sites in the EC and the striatum. Neurons with misfolded tau (Alz-50⁺) in the cell body can be found in the EC but not in the CPu, a region that, in most cases, does not develop tau pathology in AD. Notably, the amount of P301Ltau expression (approximated from GFP intensity) does not correlate with Alz-50 reactivity in the EC or CPu.