M. A. Andres

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.

Reactive gliosis of immature Bergmann glia and microglial cell activation in response to cell death of granule cell precursors induced by methylazoxymethanol treatment in developing rat cerebellum

Abstract The morphology, organization and expression of proliferating cell nuclear antigen (PCNA) and the cytoskeletal proteins vimentin and GFAP in immature Bergmann glial cells were studied after a developmental injury induced by a single dose of the cytotoxic agent methylazoxymethanol (MAM) administered on postnatal day 5. This drug, which produces cell death of cerebellar granule cell precursors, did not induce apoptosis in Bergmann glial cells, which are in a proliferative stage. After MAM treatment, PCNA staining showed a severe depletion of PCNA-positive granule cell precursors, whereas PCNA-positive Bergmann glial nuclei in the Purkinje cell layer were preserved. Moreover, the quantitative analysis revealed an increase in the density of both Purkinje cells and PCNA-positive Bergmann glial cells per mm of Purkinje cell layer in MAM-treated rats relative to age-matched controls, but the numerical ratio between these two cell populations remains invariable after MAM treatment. Vimentin and GFAP immunocytochemistry revealed a reinforcement of the Bergmann glial palisade with overexpression of both proteins and thicker immunoreactive glial processes in MAM-treated rats. At the ultrastructural level, Bergmann glial processes closely associated with dying cells in different stages of apopotosis were observed. Frequently, these processes enclosed dying cells in extracellular compartments. Furthermore, phagosomes containing apoptotic bodies were found in Bergmann fibers of MAM-treated rats. These data indicate that the cell death of granule cell precursors triggers a reactive response in immature Bergmann glia. We suggest that this response reflects the plasticity of Bergmann glia to control the neuronal microenvironment in the maturing molecular layer, protecting healthy cells against the potentially harmful contents of dying cells. In situ labeling of cell death with the TUNEL method revealed that the cell death of granule cell precursors is of the apoptotic type. The participation of ameboid microglial cells in the phagocytosis of apoptotic cells was shown with tomato lectin histochemistry and ultrastructual analysis. Moreover, the presence of mitosis in this microglial population demonstrates its proliferative activity in regions of extensive cell death.

Protein-synthesis inhibition induces perichromatin granule accumulation and intranuclear rodlet formation in osmotically stimulated supraoptic neurons

The distribution of perichromatin granules (PGs), a storage form of pre-mRNAs, was studied in supraoptic neurons of control and osmotically stimulated rats, and also after treatment with cycloheximide, a protein-synthesis inhibitor. In non-cycloheximide-treated rats, neuronal activation by dehydration significantly decreased the number of PGs. Conversely, PGs were drastically increased in the supraoptic neurons of dehydrated rats treated with cycloheximide for 4 h. This suggests that cycloheximide does not interfere with the transcriptional activation induced by dehydration, but it affects the processing of newly synthesized pre-mRNAs. Moreover, protein-synthesis inhibition was associated with the formation of intranuclear bundles of tubular filaments.

Number of nucleoli and coiled bodies and distribution of fibrillar centres in differentiating Purkinje neurons of chick and rat cerebellum.

We used differentiating chick and rat Purkinje cells to investigate in homologous neurons the influence of the number of nucleolar organizer regions (two in the chick and six in the rat) on the behaviour of the nucleolus and coiled bodies. We employed specific silver-staining methods on smear preparations and on semithin and ultrathin sections. In chick Purkinje cells the number of nucleolar silver-staining granules increased from 15.7±3 (mean±SD) at embryonic day 13 to 23.8±3 at post-hatching day 7. These nucleolar granules were unevenly distributed between the two nucleoli of binucleolated cells. Electron-microscopic cytochemistry showed that nucleolar granules are equivalent to the fibrillar centres with their associated shell of dense fibrillar component. A reduction in the number of nucleoli was found during the differentiation of both chick and rat Purkinje cells, although in mature cells the average number of nucleoli per cell was higher in the chick (1.60) than in the rat (1.07). The number of coiled bodies decreased from 1.33 in newborn rats to 0.47 at postnatal day 90 in the rat. Coiled bodies were not observed in homologous chick Purkinje cells. The dynamic behaviour of nucleoli and coiled bodies during neuronal differentiation and the relationship of these two nuclear organelles with the number of nucleolar organizer regions is discussed.

Effects of cycloheximide on the structural organization of the nucleolus and the coiled body in normal and stimulated supraoptic neurons of the rat

SummaryThis study was designed to determine the effects of cycloheximide, a protein synthesis inhibitor that interferes with rRNA synthesis and processing, on the nucleoli and coiled bodies of supraoptic nucleus neurons from normally-hydrated and osmotically-stimulated rats. The number of nucleoli and the nucleolar size were estimated on smear preparations of previously silver-impregnated supraoptic nucleus. No significant differences were registered in the mean number of nucleoli per cell in cycloheximide-treated rats. The number of nucleoli per neuron remained constant, at about 1.3, in all animal groups, suggesting that the nucleoli number is strictly regulated in differentiated neurons. By contrast, a significant reduction in the average nucleolar volume of supraoptic nucleus neurons was detected in cycloheximide-treated groups of rats in comparison with their equivalent non-treated groups. By electron microscopy, most nucleoli and coiled bodies of supraoptic nucleus neurons exhibited cycloheximide-induced alterations in their fine structure and configuration. Nucleolar changes included the occurrence of a few large fibrillar centres, the formation of microspherules and small intranucleolar vacuoles or dilated interstices, and the partial segregation of nucleolar components coupled with the transformation of reticulated nucleoli — a nucleolar configuration characteristic of supraoptic nucleus neurons of non-cycloheximide-treated rats — into compact ones. The redistribution of nucleolar components might reflect the interference with rDNA transcription, and also supports the hypothesis that the normal assembly of these components into the nucleolus depends upon ongoing nucleolar transcription. Concerning coiled bodies, most of them revealed ultrastructural alterations, particularly segregation of the amorphous matrix, compactation of coiled threads and formation of coiled body-derived dense bodies of fibrillar nature. Moreover, cycloheximide also induced the formation of smaller dense bodies — here referred to as dense microbodies — which presumably represent a distinct nuclear entity different from coiled bodies. Ultrastructural silver staining of nuclear bodies showed a selective silver reaction on the dense fibrillar component of normal and altered coiled bodies, as well as on the dense microbodies. The possible relationship between the nucleolus and both coiled bodies and dense microbodies is discussed.

Schwann cell nuclear remodelling and formation of nuclear and coiled bodies in Guillain-Barré syndrome

Abstract We have examined the reorganization of the cell nucleus in myelin-related Schwann cells (SCs) in a case of acute Guillain-Barré syndrome (GBS). Spinal root samples of the GBS case and human controls were processed for light and electron microscopy. The cytochemical EDTA method for ribonucleoproteins (RNPs) and a specific silver staining technique for nucleolar organizer regions were used on ultrathin sections. In SCs of the GBS case, we observed a significant increase in nuclear size (64.99 ± 10.47 μm2 in the GBS vs 35.07 ± 8.74 μm2 in the controls mean ± SD) accompanying partial decondensation of heterochromatin domains and elaboration of an extensive network of RNP-containing perichromatin fibrils. In addition, the formation of two types of nuclear structures, coiled bodies and nuclear bodies of Bouteille, was induced in SCs of the case of acute GBS. Free coiled bodies were observed in the nucleoplasm and were characteristically stained with both RNP and silver procedures. Typical “simple” and “complex” nuclear bodies were regularly found, sometimes in association with coiled bodies. On the basis of cell nucleus physiology, all of these changes are considered cytological indicators of enhanced transcription and cellular hyperactivity, and they seem to reflect a reactive response of SCs triggered by the constellation of cellular and humoral signals associated with acute GBS.

Apolipoprotein E expression in the cerebellum of normal and hypercholesterolemic rabbits.

We have studied the expression of apolipoprotein E (ApoE) mRNA in the cerebella of control and experimental rabbits fed with a cholesterol-rich diet for 8 weeks. Cholesterol-treated rabbits show a dramatic increase in serum cholesterol levels; however, no significant variations in the expression level of cerebellar ApoE mRNA were found in comparison to control rabbits. In addition, no differences were observed between control and hypercholesterolemic rabbits in the in situ hybridization pattern of ApoE mRNA on cerebellar cortex sections. ApoE mRNA was localized in astroglial processes associated with Purkinje cell bodies and dendrites, granule cell clusters, blood vessels and nerve fibers of the white matter. No expression of ApoE mRNA was observed in Purkinje and granule cell neurons. Polarized light examination of cryostat cerebellar sections revealed the absence of cholesterol-rich microglia/macrophage cells induced by the hypercholesterolemia. In this way, neither reactive microglial cells nor perivascular phagocytes were found by ultrastructural analysis in hypercholesterolemic conditions. The pattern of glial fibrillary acidic protein of the astroglial cells of the cerebellar cortex as well as their nuclear size were unchanged following cholesterol treatment, indicating the absence of astroglial activation induced by hypercholesterolemia. Our results suggest that cerebellar ApoE does not contribute to the general cholesterol homeostasis outside of the brain and supports the view that this cerebellar ApoE is involved in paracrine and autocrine functions particularly related with synapse turnover and membrane remodelling of astroglial cells.