Accumulation of nuclear DNA damage or neuron loss: molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

Abstract

According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.

Fig. 1

Principles of in situ nick translation (ISNT) (a) and measuring UDS with autoradiography (b). For ISNT (a) tissue sections are incubated with a reaction solution containing buffer, MgCl₂, 2-mercaptoethanol, endonuclease-free E. coli DNA polymerase I, dATP, dGTP, dCTP and [³H]dTTP. The reaction is terminated by washing the slides with buffer. Afterwards sections are Feulgen-stained (which also removes all [³H] activity not incorporated into the nDNA; [–162]), dipped into liquid photoemulsion, exposed, and developed. Poststaining can be performed with hematoxyline among other standard histologic stains. For measuring UDS with autoradiography (b) animals are injected with [³H]TdR and sacrificed 1 or 2 h later. Tissue sections are then also Feulgen-stained, dipped into liquid photoemulsion, exposed, developed, and poststained. Further details can be found in [39].

Fig. 2

Autoradiographic detection of nDNA single-strand breaks in neurons in the thalamus of a 12-month-old mouse (a and b) and a 28-month-old mouse (c) with in situ nick translation (ISNT) (Brasnjevic et al., unpublished results; the corresponding experiments were described in detail in [10]). (a) Background signal in the autoradiographs after performing ISNT without use of E. coli DNA polymerase I. No silver grains were found after 5 days of exposure. (b and c) Autoradiographic signal after performing ISNT with E. coli DNA polymerase I. Note the clearly visible age-related increase in the number of silver grains over the nuclei in the investigated brain region, indicating an age-related increase in the relative amount of nDNA single-strand breaks. The scale bar represents 15 μm.