Induction of tau pathology by intracerebral infusion of amyloid-beta -containing brain extract and by amyloid-beta deposition in APP x Tau transgenic mice

Abstract

Alzheimer's disease presents morphologically with senile plaques, primarily made of extracellular amyloid-beta (A beta) deposits, and neurofibrillary lesions, which consist of intracellular aggregates of hyperphosphorylated tau protein. To study the in vivo induction of tau pathology, dilute brain extracts from aged A beta-depositing APP23 transgenic mice were intracerebrally infused in young B6/P301L tau transgenic mice. Six months after the infusion, tau pathology was induced in the injected hippocampus but also in brain regions well beyond the injection sites such as the entorhinal cortex and amygdala, areas with neuronal projection to the injection site. No or only modest tau induction was observed when brain extracts from aged nontransgenic control mice and aged tau-depositing B6/P301L transgenic mice were infused. To further study A beta-induced tau lesions B6/P301L tau transgenic mice were crossed with APP23 mice. Although A beta deposition in double-transgenic mice did not differ from single APP23 transgenic mice, double-transgenic mice revealed increased tau pathology compared to single B6/P301L tau transgenic mice predominately in areas with high A beta plaque load. The present results suggest that both extract-derived A beta species and deposited fibrillary A beta can induce the formation of tau neurofibrillary pathology. The observation that infused A beta can trigger the tau pathology in the absence of A beta deposits provides an explanation for the discrepancy between the neuroanatomical location of A beta deposits and the development and spreading of tau lesions in Alzheimer's disease brain.

Figure 1

Tau pathology in heterozygous P301L tau transgenic mice on the genetic C57BL6/J background (B6/P301L). A and F: Overview picture taken from an AT8-stained section shows the distribution of tau pathology in female B6/P301L mice at 12 months (A) and 21 months (F) of age. B–E and G–J are high-magnification pictures taken from A and F and reveal in the 21-month-old mice but not the 12-month-old mice a substantial amount of tau lesions in the hippocampus (B, G; top middle box in A and F), lateral entorhinal cortex (C, H; bottom right box in A and F), amygdala (D, I), and deep mesencephalic nucleus (E, J; top left box in A and F). Insets in I and J show Gallyas-positive tangles, indicating that a large subset of the accumulating tau proteins form insoluble aggregates. All panels have same magnification. Scale bar = 100 μm.

Figure 2

Histological and biochemical analysis of the injected brain extracts. A: Aβ-immunostaining of neocortex of a 24-month-old APP23 transgenic mouse, similar to the one from which the extract was prepared, reveals extensive cerebral amyloid deposition. B: No amyloid was found in a nontransgenic age-matched littermate control brain. C: AT8-immunostaining of the brainstem of a female 21-month-old B6/P301L transgenic mouse, similar to the one from which the tau extract was prepared, reveals numerous tau-positive deposits. All panels have the same magnification. D: Left: Immunoblotting with antibody 6E10 specific to human Aβ. Lane 1, synthetic Aβ1-40 and 1-42 (1 ng/μl each); lane 2, APP23 brain extract; lane 3, nontransgenic APP23 littermate control. Right: Western blotting analysis with HT7 antibody specific to human tau. Lane 1, AD brain extract reveals the various human tau isoforms (arrowheads); lane 2, B6/P301L brain extract shows the transgenic 4R0N human isoform. Scale bar = 50 μm.

Figure 3

Intracerebral injection of Aβ-containing mouse brain extract induces tau pathology in the hippocampus of B6/P301L tau transgenic mice. Young 5- to 6-month-old B6/P301L transgenic mice and control littermates were injected unilaterally in the hippocampus and overlying neocortex with various brain extracts and analyzed 6 months later. A: Overview picture taken from an AT8-stained section shows the pattern of induced tau deposits throughout the injected hippocampus but also entorhinal cortex (asterisk) of a B6/P301L transgenic mouse injected with the APP23 brain extract (the injected site is the right one in the panel). B: The injected hippocampal area, box in A, is shown in higher magnification. C: Injection with the nontransgenic control extract did not induce tau pathology in the hippocampus. D and E: Modest induction of tau pathology was seen with the B6/P301L extract (D), whereas no induced tau deposits were observed after PBS injection (E). F: When Aβ-containing APP23 extract was injected into a nontransgenic B6/P301L mouse, no tau lesions were induced. Scale bar = 100 μm.

Figure 4

Tau pathology induction in various regions in B6/P301L tau tg mice. A: Stereological analysis of percent area occupied by AT8-positive staining (two-way analysis of variance, extract × hemisphere) in the hippocampus (HPS) confirms the significant induction of tau deposition by the APP23 extract compared to PBS (⁺n = 5/group; P < 0.0001; Newmann-Keuls posthoc tests; indicated is SEM) and compared to the contralateral site (*P < 0.0001). Tau induction also occurred in some brain regions away from the injection site, such as the entorhinal cortex (ENT) (B) and the amygdala (AMG) (C). Analysis again revealed significant induction of the APP23 extract compared to PBS (⁺n = 5/group, P < 0.0001) and to the contralateral site (*P < 0.05), which for the amygdala also showed some induction compared to PBS (P < 0.001). D and E: No induced tau pathology was found in the sensorimotor cortex (CTX) (D) and brain stem (BS) (E) (n = 5/group; P > 0.05). APP23, extract from an aged APP23 mouse brain; WT, wild-type nontransgenic extract; P301L, extract from an aged B6/P301L mouse brain.

Figure 5

Induced tau lesions in B6/P301L × APP23 mice. A and B: AT8-stained coronal sections show tau pathology in the entorhinal cortex and adjacent CA1 field of an aged male 28-month-old double-transgenic B6/P301L × APP23 mouse (A) whereas no pathology was observed in a male age-matched B6/P301L mouse (B). C: Stereological analysis of tau immunoreactivity (AT8 antibody) in the entorhinal cortex (ENT), hippocampus (HPS), and amygdala (AMG) revealed tau induction in B6/P301L × APP23 transgenic animals (n = 7) compared to both single transgenic B6/P301L mice (n = 7) and to APP23 littermates (n = 4) (one-way analysis of variance for genotype: *ENT, P < 0.01; HPS, P < 0.05; AMG, P < 0.001; indicated is SEM) but not in the brain stem. D: Stereological analysis of amyloid load in the neocortex (CTX), hippocampus (HPS), amygdala (AMG), and brain stem (BS) revealed no difference between the double-transgenic mice and the single APP23 transgenic littermates in any of the brain region analyzed (P > 0.05). E–H: High magnification of an adjacent section to A double immunostained for tau (AT8) and Aβ shows the distribution of tau pathology in close vicinity of amyloid deposits in the entorhinal cortex (E), and hippocampus (F) (see also boxed areas in A). In the piriform cortex (G), and dorso- and ventrolateral amygdala (asterisks in H), no significant induction of tau pathology was observed despite robust amyloid load. Notably in the basolateral part of the amygdala, substantial induction of tau lesions were observed (arrowheads in H) although no significant amyloid deposition was apparent in this area. E–G have the same magnification. I: Gallyas silver staining shows that many of the induced tau deposits in the entorhinal cortex were of fibrillary nature. Scale bars = 75 μm.