Apoptosis induced by methylazoxymethanol in developing rat cerebellum: organization of the cell nucleus and its relationship to DNA and rRNA degradation

Abstract

We present a cytological and biochemical study of the cell death of granule cell precursors in developing rat cerebellum following treatment with the cytotoxic agent methylazoxymethanol (MAM) during the first postnatal week. The density of apoptotic figures per square millimeter progressively increases after 6, 12, 24 and 44 h of treatment, whereas cells immunoreactive for proliferating cell nuclear antigen tend to disappear in the external granular layer (EGL). DNA migration on gel electrophoresis reveals a typical ladder pattern of internucleosomal cleavage following MAM treatment, whereas gel electrophoresis of rRNA shows a conspicuous degradation of both 28S and 18S rRNAs. Ultrastructural analysis has revealed the alterations of structures containing chromatin and ribonucleoprotein (RNP) in dying cells of the EGL. The typical granular beaded configuration of the condensed chromatin changes to a denser, more homogeneous texture suggesting nucleosomal disruption. The reorganization of RNP nuclear domains is reflected by the appearance of dispersed nucleoplasmic RNP particles and the formation of a coiled-body-like structure. However, typical nuclear domains involved in the splicing of RNAs, namely interchromatin granule clusters and typical "coiled bodies", are not found in apoptotic cells. Intranuclear bundles of filaments have also been detected. In the cytoplasm, the presence of dispersed single ribosomes is an initial sign of apoptosis. The massive dispersion and disruption of ribosomes detected after 24 h and 44 h of MAM treatment is reflected by the degradation of both 28S and 18s rRNAs. These results show that MAM treatment provides a useful experimental model for the study of apoptosis in the developing central nervous system. The organization of the cell nucleus in cells undergoing apoptosis clearly reflects a disruption of the nuclear compartments involved in transcription and the processing and transport of RNA and is related to the patterns of DNA and rRNA degradation.

Rearrangement of nuclear ribonucleoprotein (RNP)‐containing structures during apoptosis and transcriptional arrest

“…A rearrangement of RNPs in cells of the developing rat cerebellum has been described by Lafarga and coworkers (Lafarga et al, 1997) following treatment with the cytotoxic (arrows) is densely packed and forms spherical areas typical of nucleolar segregation. e) gold immunolabeling for DNA on the condensed chromatin (C); the nucleolus (asterisk) is surrounded by and intermingled with nucleoplasmic RNPs (arrowheads).…”

“…A rearrangement of RNPs in cells of the developing rat cerebellum has been described by Lafarga and coworkers (Lafarga et al, 1997) following treatment with the cytotoxic Clevenger and Epstein, 1984) after incubation with an undiluted MoAb (ICN Pharmaceuticals, Italy), and with an Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes). a) control cells; b,c) cells treated with 20 µM DRB for 5 h and for 24 h, respectively: the discretely dotted immunolabeling of controls becomes progressively more diffuse with increasing treatment time.…”

“…As a consequence of this proteolytic process, the partial cleavage and/or the ectopic association of different protein components is capable of enhancing the immunogenic potential of these proteins. Moreover, HERDS have also been shown to contain RNA, as detected by cytochemical techniques using light and electron microscopy (Biggiogera et al, 1998).A rearrangement of RNPs in cells of the developing rat cerebellum has been described by Lafarga and coworkers (Lafarga et al, 1997) following treatment with the cytotoxic (arrows) is densely packed and forms spherical areas typical of nucleolar segregation. e) gold immunolabeling for DNA on the condensed chromatin (C); the nucleolus (asterisk) is surrounded by and intermingled with nucleoplasmic RNPs (arrowheads).…”

Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair

“…They frequently appeared detached from the nuclear envelope and exhibited a fine granular texture (Fig. 2E), corresponding to the cross-section of closely packed chromatin fibers (32).…”

Caspase-dependent cleavage of nucleic acids

“…59,61,62 However, recent reports revealed that both the 28S rRNA and the 18S rRNA are cleaved in several cell lines during apoptosis. 63 Interestingly, it has also been reported that the mitochondrial 16S rRNA molecule can be cleaved after apoptotic stimulation. 64 The apoptotic cleavage sites in rat 28S rRNA have been mapped to the two largest divergent domains, D2 (approx.…”