In vivo imaging reveals dissociation between caspase activation and acute neuronal death in tangle-bearing neurons

Abstract

Accumulation of neurofibrillary tangles (NFTs) in Alzheimer's disease correlates with neuronal loss and cognitive decline, but the precise relationship between NFTs and neuronal death and downstream mechanisms of cell death remain unclear. Caspase cleaved products accumulate in tangles, implying that tangles may contribute to apoptotic neuronal death. To test this hypothesis, we developed methods using multiphoton imaging to detect both neurofibrillary pathology and caspase activation in the living mouse brain. We examined rTg4510 mice, a reversible mouse model of tauopathy that develops tangles and neuronal loss. Only a small percentage of imaged neurons were caspase activity positive, but the vast majority of the cells with active caspases contained NFTs. We next tested the hypothesis that caspase activation led to acute, apoptotic neuronal death. Caspase positive cell bodies did not degenerate over hours of imaging, despite the presence of activated executioner caspases. Suppression of the transgene, which stops ongoing death, did not suppress caspase activity. Finally, histochemical assessments revealed evidence of caspase-cleaved tau, but no TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) positive or apoptotic nuclei. With the novel technique of observing NFTs and caspase activation in the living brain, we demonstrate that aggregated tau in neurons can be associated with caspase activation, but that caspase activation is not sufficient to cause acute neuronal death in this model.

Figure 1.

ThioS labels neurofibrillary tangles in vivo. Topical application of 0.025% ThioS to the cortex of rTg4510 mice was used to observe neurofibrillary tangles. a, b, Low-magnification micrographs show that NFTs are prevalent throughout the cortex of 6-month-old (a) and 9-month-old (b) mice. c–e, At high resolution, most of these lesions appear blue-green and fibrillar although occasionally less fibrillar lesions are observed which are more blue in color (arrow). ThioS positive neurons were not observed in control animals (data not shown). Scale bars: a, b, 20 μm; c–e, 10 μm.

Figure 2.

Caspase activation occurs in rTg4510 cortex. a, b, e–h, An indicator of polycaspase activation applied in vivo (red) shows that activated caspases are present both in cells with ThioS positive tangles (green-blue; arrows) and without (arrowheads). Rarely, cells with active caspases exhibit nuclear fragmentation (asterisks), a morphological characteristic of apoptotic neurons. c, d, Indicators specific to caspase 3 and 7 activation (red; c) and to caspase 8 (green; d) label neurons showing that both initiator and executioner caspases are active in these mice. e, Pretreatment with the pan-caspase inhibitor z-vad-fmk prevented caspase activation although ThioS positive cells are still visible. f, After washing out the inhibitor, caspase activation reappears, usually in tangle-bearing cells as expected. g, h, Transgene suppression for 2 weeks does not prevent caspase activation. Scale bar, 10 μm.

Figure 3.

Histological and molecular evidence of caspase upregulation. a, Immunostaining for tau cleaved at Aps 421/422 revealed cell bodies (arrows) and processes (arrowheads) in the hippocampus and cortex of rTg4510 tau transgenic mice. b, This activation persisted after transgene suppression with doxycycline (dox). c, No staining was observed in nontransgenic control mice. d, Caspase 8 mRNA levels (normalized to GAPDH) are elevated in rTg4510 mice at 5.5 and 8.5 months compared with control levels (dotted line). Transgene suppression does not significantly alter caspase levels. *p < 0.05, Mann–Whitney U test. Scale bar, 20 μm.

Figure 4.

Caspase activation does not lead to rapid degeneration and persists after transgene suppression. a, In untreated rTg4510 animals, caspase positive cell bodies (red) and cells with NFTs (blue-green) remain stable over the course of hours of imaging. b, c, Caspase positive cells remain even after transgene suppression and these cell bodies similarly do not degenerate over the course of hours. The black arrowhead indicates a cell with activated caspases but no tangle, white arrowheads show cells with both NFTs and activated caspases, and the black arrow shows a cell with NFTs but no active caspases. Scale bar, 10 μm.