Despite years of study, the mechanisms of neuron loss in AD are still incompletely understood. Apoptosis has been proposed as a potential mechanism leading to the neuronal death observed in this devastating neurodegenerative disorder (reviewed in [34–36]). Here, in the 5XFAD amyloid plaque mouse model, we have demonstrated progressive loss of neurons that correlates strongly with both intraneuronal Aβ42 and Caspase-3 activation. We observe a time course in 5XFAD mice in which intraneuronal Aβ42 first appears at 1.5 months of age in large pyramidal neurons of Layer 5 cortex and subiculum, concomitant with the observation of activated Caspase-3 in these neurons, but prior to amyloid deposition. By 4 months of age, 5XFAD mice exhibit amyloid plaques and the appearance of Caspase-3 positive puncta in Layer 5 cortex and subiculum that suggest neurodegeneration, but at a time when statistically significant neuron loss is not yet evident. At 6 months of age, 5XFAD mice as a whole show a trend toward neuron loss, in which Layer 5 neurons appear to split into two subgroups, those that display neuron loss and those that do not. However, by 9 months, neuron loss is statistically significant in Layer 5 cortex of 5XFAD mice. This time course, although correlative, suggests the possibility that intraneuronal Aβ42 triggers Caspase-3 activation, which in turn induces apoptosis-mediated neurodegeneration and eventual neuron loss in 5XFAD mice.
The 5XFAD mouse, along with the APPSLPS1KI, is one of a few known mouse models that exhibits significant neuron loss in addition to displaying other AD hallmarks such as amyloid plaques [10, 11, 16]. Our unbiased stereological counting of 5XFAD mice revealed a significant decline in large pyramidal neurons of the Layer 5 cortex starting at 9 months of age. Our data is consistent with previous studies, except we have demonstrated that significant 5XFAD neuron loss begins at an earlier age than previously reported . Interestingly, a division develops at 6 months that appears to separate 5XFAD mice into those that exhibit significant neuron loss and those that have yet to surpass the point where neuron loss is occurring at a measurable rate. The ~6 month age period marks the first appearance of oligomeric N-terminally truncated Aβ peptides with pyroglutamate and an increasing decline of motor abilities in 5XFAD mice [16, 23].
Consistent with previous studies [10, 32], we observed intraneuronal Aβ42 as small puncta located in neuronal soma of 5XFAD mice. The intraneuronal Aβ42 became progressively more difficult to detect in 5XFAD sections after 1.5 months of age, presumably because highly concentrated Aβ42 plaques effectively blocked visualization of smaller and less prominent intraneuronal Aβ42 puncta. Despite this difficulty, 5XFAD mice exhibited a subset of neurons that harbored intraneuronal Aβ42 past 4 months. The early appearance of intraneuronal Aβ42 and its correlation with neuron loss in 5XFAD mice is also shared with the APPSLPS1KI transgenic mouse model [11, 33, 37, 38].
We found that intraneuronal Aβ42 in 5XFAD mice was partially co-localized to endosomes and lysosomes, as indicated by co-labeling for Transferrin receptor and LAMP1, respectively. This corresponded well with previous findings that intraneuronal Aβ42 localizes to late endosomal multivesicular bodies  or lysosomes ; the latter observation is particularly significant, as it was obtained in the 5XFAD mouse. Intraneuronal Aβ42 localization to endosomes and lysosomes is also supported by previous studies that have proposed a mechanism in which the ubiquitin-proteasome system is disrupted, causing impaired protein degradation and increased Aβ accumulation [39, 40].
It has been hypothesized that apoptosis is involved in the neuron loss seen in AD (reviewed in [34–36]). For example, activated Caspase-3 immunoreactivity has been reported to be present in AD brain [41, 42] and in APP transgenic mice [43, 44]. Interestingly, Aβ42 has been shown to cause cytochrome c release from mitochondria , which activates Caspase-3 and induces apoptosis, thus providing a potential mechanism for intraneuronal Aβ42-mediated neuron loss in 5XFAD mice.
In 5XFAD mice, activated Caspase-3-positive puncta were present throughout Layer 5 cortex and subiculum, often in close proximity to Aβ42-positive plaques. Caspase-3 activation was not apparent in both aged matched non-transgenic littermate and 5XFAD; BACE1−/− mice. We speculate that these activated Caspase-3-positive puncta are likely the remnants of neurons that died by apoptosis, a conclusion supported by the observation of co-localized activated Caspase-3 and neuron-specific class III β-tubulin (Figure 6F). The Caspase-3 puncta in 5XFAD brain are reminiscent of activated Caspase-3-positive AD neurons that exhibit typical features of apoptosis such as condensed cytoplasm and shrunken nuclei [46, 47]. In addition, the association of activated Caspase-3 and neuron loss in 5XFAD mice is consistent with the findings of apoptotic neuron death and “apoptotic bodies” found in a different PS/APP (PS1M146L and K670N/M671L) mouse model of AD . While not always coexistent, we observed large pyramidal neurons from 1.5 month-old 5XFAD mice that contained both punctate intraneuronal Aβ42 within the neuron soma and activated Caspase-3 throughout the cell body and proximal dendrites (Figure 7K, L). We speculate that these neurons could represent an early stage of apoptotic neuronal degeneration and death. Interestingly, a recent study found activated caspase-3 in dendritic spines of hippocampal neurons in the Tg2576 APPswe transgenic mouse model , supporting the dendritic localization of activated Caspase-3 that we have observed in 5XFAD mice.