Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Evidence for apoptotic cell death

Abstract

Neuronal loss is prominent in Alzheimer's disease (AD), and its mechanisms remain unresolved. Apoptotic cell death has been implicated on the basis of studies demonstrating DNA fragmentation and an up-regulation of proapoptotic proteins in the AD brain. However, DNA fragmentation in neurons is too frequent to account for the continuous neuronal loss in a degenerative disease extending over many years. Furthermore, the typical apoptotic morphology has not been convincingly documented in AD neurons with fragmented DNA. We report the detection of the activated form of caspase-3, the central effector enzyme of the apoptotic cascade, in AD and Down's syndrome (DS) brain using an affinity-purified antiserum. In AD and DS, single neurons with apoptotic morphology showed cytoplasmic immunoreactivity for activated caspase-3, whereas no neurons were labeled in age-matched controls. Apoptotic neurons were identified at an approximate frequency of 1 in 1100 to 5000 neurons in the cases examined. Furthermore, caspase-3 immunoreactivity was detected in granules of granulovacuolar degeneration. Our results provide direct evidence for apoptotic neuronal death in AD with a frequency compatible with the progression of neuronal degeneration in this chronic disease and identify autophagic vacuoles of granulovacuolar degeneration as possible means for the protective segregation of early apoptotic alterations in the neuronal cytoplasm.

Figure 1.

Activation of caspase-3 in AD and DS. a: Activated caspase-3 in apoptotic hippocampal granule cells in pontosubicular neuron necrosis. b: Subicular neuron of case DS 1 immunoreactive for activated caspase-3 with a condensed nucleus and shrunken cytoplasm indicative of apoptotic cell death. c: CM1-positive neuron in layer 2 of temporal isocortex in case AD 9. d: CM1-positive subicular neuron in case DS 1 displaying a condensed nucleus and cytoplasm. e and f: CA1 neurons of case DS 3; granules of GVD are immunolabeled with the CM1 antibody against activated caspase-3 (arrowheads), whereas other cytoplasmic compartments remain unstained. g: CA1 neuron in case DS 3 double-labeled with AT8; a small tangle (arrowhead) and diffuse AT8 positivity in a neuron with CM1-positive GVD (arrow). Activated caspase-3 is depicted in brown (DAB), AT8 in blue (Fast Blue). Nuclear counterstaining with hematoxylin, except for g, where no counterstaining was performed.Original magnifications, ×1000 (a-d, f, g) and ×400 (e).

Figure 2.

Frequency of GVD in hippocampal neurons with and without AT8 positive cytoskeletal alterations (neurofibrillary tangles and pretangle changes). CA1 and subicular neurons were analyzed in cases AD 1–8 and DS 1–4. Among AT8-positive neurons, 25.99 ± 5.25% were found to contain GVD as compared to 4.69 ± 1.47% of AT8-negative neurons (P = 0.0012; Mann-Whitney U test), and 78.25 ± 7.76% of GVD was found in AT8-positive neurons.