Elsevier

Brain Research

Volume 849, Issues 1–2, 4 December 1999, Pages 67-77
Brain Research

Research report
DNA strand breaks in Alzheimer's disease

https://doi.org/10.1016/S0006-8993(99)02004-1Get rights and content

Abstract

The goal of this study was to investigate the presence of DNA damage in Alzheimer's disease (AD) utilizing independent assays for three different types of DNA strand breaks. Sections from hippocampi of AD brains, brains with Alzheimer neurofibrillary changes (Ch) from non-demented individuals, and controls (C) were labeled with (1) the TUNEL assay to identify blunt-ended and 3′ protruding termini of breaks in double-stranded DNA, (2) the Klenow assay to detect single-stranded and double-stranded breaks with protruding 5′ termini, and (3) the Apostain assay which utilizes a monoclonal antibody to single-stranded DNA and is based on the decreased stability of apoptotic DNA to thermal denaturation caused by DNA breaks. The highest incidence of nuclei positive for either molecular form of DNA strand breaks was detected in AD, followed by Ch, and controls (C). In either AD and Ch, the incidence of TUNEL- or Klenow-positive nuclei did not differ significantly, but was higher than the incidence of Apostain-positive nuclei. With all three assays, the highest incidence of positive nuclei was in the molecular layer of CA1. In the majority of nuclei positive for either the Klenow or the Apostain assay, the product of the labeling reaction was localized either to the periphery of the nucleus or to distinct clumps of chromatin (or both). With the TUNEL assay, the majority of positive nuclei were diffusely labeled. In both AD and Ch, the individual positive nuclei were labeled with both the Klenow and the TUNEL assays. The results indicate high incidence of nuclei with either double-stranded or single-stranded DNA breaks in AD, which, for the forms detectable with the Klenow or TUNEL assays, were colocalized.

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.

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