Research reportDNA strand breaks in Alzheimer's disease
Introduction
Alzheimer's disease (AD) is a devastating degeneration which accounts for half to two-thirds of all cases of late-life intellectual failure in many developed countries where life expectancies are high [45]. AD represents the fourth leading cause of death in the United States and its profound morbidity has a major socioeconomic impact [40]. The disease is characterized by progressive dementia associated with neuropathological findings of neurofibrillary tangles and the senile plaques [19]. Functionally, the disease causes synaptic and neuronal loss, which strongly correlate with the degree of cognitive impairment 34, 43, 54.
The etiopathogenesis of AD is poorly understood. One of the mechanisms responsible for cellular death in AD might be an accumulation of DNA damage (strand breaks). Utilizing biochemical studies on isolated nuclei, Mullaart et al. [38] found at least a twofold higher level of DNA damage in cortex from individuals with AD as compared to C. Biochemical methods, however, have limited value in the evaluation of histopathological material and provide only a global index of DNA damage. Therefore, for the purpose of detecting DNA damage on a single cell level, several in situ methods have been developed. These assays enzymatically attach labeled nucleotides (with biotin, digoxigenin, or a fluorophore) at the 3′ ends of DNA strand breaks. The TUNEL assay (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling [15]) utilizes the enzyme terminal deoxynucleotidyl transferase to label both 3′ protruding and blunt-ended termini of double-stranded DNA (in the presence of Co2+) [20]. A similar method, the Klenow assay (also known as the in situ nick translation, ISNT [18]), is based on the same principle, but uses the Klenow enzyme (large fragment of DNA polymerase I) to label only single-stranded breaks (nicks) with 5′ overhangs of double-stranded DNA and minimally label 3′ protruding and blunt-ended termini [20]. A novel approach, Apostain assay, utilizes a monoclonal antibody to single-stranded DNA and is based on the decreased stability of DNA containing strand breaks during thermal denaturation. Initial experience with this method in non-neuronal preparations indicates that it may to be both more specific and more sensitive to detect early stages of apoptosis than the commonly used TUNEL assay [13].
The studies of DNA damage in neuropathological material concentrated, so far, nearly exclusively on the detection of the apoptotic form of DNA damage, characterized biochemically by internucleosomal double-stranded DNA breaks and histopathologically by a positive reaction with the TUNEL assay. The presence of TUNEL-positive cells was demonstrated in AD 2, 11, 24, 25, 31, 35, 46, 49, 50, 51, 52, 53, 57, Huntington disease 5, 41, 55, Parkinson's disease 23, 37, 56, brain trauma 9, 27, 42, and hypoxia/ischemia 7, 21, 28. No studies have yet investigated the presence of either the Klenow- or the Apostain-positive types of DNA strand breaks in neurodegenerative diseases. Since the presence of different molecular forms of DNA damage might have different biological significance and might also point to potentially different etiopathogenic mechanisms, the goal of this study was to extend the investigation of DNA damage in AD by simultaneously assaying for all three distinct molecular forms of DNA strand breaks. The first objective was to determine whether or not there is a significant difference in the incidence and regional distribution of nuclei with different forms of DNA strand breaks in AD brain. The second objective was to compare the staining patterns of the positive nuclei with special emphasis on the detection of the reaction product on the periphery of the nucleus or in distinct clumps (consistent with apoptotic nuclear morphology). The third objective was to investigate whether or not different molecular types of DNA strand breaks are present in individual nuclei.
Section snippets
Tissue samples
The liquid nitrogen vapor frozen blocks [59] of hippocampus from five brains with pathologically confirmed AD [22], five with Alzheimer neurofibrillary changes (Ch), and four brains from individuals without neurological abnormalities and without Alzheimer changes were obtained from Harvard Brain Tissue Resource Center (McLean Hospital, Belmont, MA). Cryostat sections (6 μm) were attached on silane-coated slides and stored at −80°C until use.
Assays for DNA strand breaks
The TUNEL assay [15] was performed according to the
Results
The goal of this study was to investigate the presence of different molecular forms of DNA damage in AD utilizing independent assays for three different types of DNA strand breaks. The study was performed on cryostat sections of hippocampus from five cases with pathologically confirmed AD, five cases with Ch, and four C without Alzheimer changes in the following anatomical regions: granule cell layer of the dentate gyrus (DG), CA4, the molecular (CA1mol), the pyramidal (CA1pyr), and the
Discussion
This study demonstrated the presence of a considerable number of nuclei with different molecular forms of DNA strand breaks in the hippocampal formation of individuals with AD and, to a lesser extent, in cases with Alzheimer neurofibrillary pathology. In agreement with previous studies in AD, we have detected nuclei positive for the TUNEL end-labeling protocol, indicating the presence of either blunt-ended double-stranded DNA breaks and/or breaks with protruding 3′ termini. An important novel
Acknowledgements
We acknowledge the assistance of Ms. H. Goolsby and Ms. W. Hobbs with the preparation of tissue sections. We would also like to thank Dr. F.M. Benes, McLean Hospital, for the use of her imaging equipment. This study is supported by grants (AG000764 and AG05134) from the National Institute on Aging. The Harvard Brain Tissue Resource Center is supported in part by PHS grant MH/NS 31862.
References (61)
- et al.
Trinucleotide (CAG) repeat length is positively correlated with the degree of DNA fragmentation in Huntington's disease striatum
Neuroscience
(1998) - et al.
Monoclonal antibody tosingle-stranded DNA is a specific and sensitive cellular marker of apoptosis
Exp. Cell Res.
(1996) - et al.
In situ detection of fragmented DNA (TUNEL assay) fails todiscriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note
Hepatology
(1995) DNA strand breaks in rat tissues as detected by in situ nick translation
Exp. Cell Res.
(1986)- et al.
Neuronal apoptosis and necrosis following spinal cord ischemia in the rat
Exp. Neurol.
(1997) - et al.
Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells
Brain Res.
(1996) - et al.
Single stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in the gerbil hippocampus
Neurosci. Lett.
(1998) - et al.
Histochemical detection of apoptosis in Parkinson's disease
J. Neurol. Sci.
(1996) - et al.
Increased levels of DNA breaks in cerebral cortex of Alzheimer's disease patients
Neurobiol. Aging
(1990) - et al.
Reactive oxygen species and Alzheimer's disease
Biochem. Pharmacol.
(1997)