Subcallosal striations: early findings of multiple sclerosis on sagittal, thin-section, fast FLAIR MR images

Since Bcl-2 protects a variety of cell types from programmed cell death, whereas Bax promotes apoptosis, the present study examines Bcl-2 and Bax proteins, and bcl-2 and bax mRNA expression in the developing cerebellum of the rat following methylazoxymethanol (MAM) acetate administration by using immunohistochemistry, Western blotting and Northern blotting. Bcl-2 expression in the developing cerebellum is observed in proliferating and differentiating cells, whereas Bax expression is higher in differentiating cells than in proliferating cells during development. Administration of MAM (0.05 microliter/g, i.p.) at postnatal day 3 produces apoptotic cell death, as detected by the characteristic morphology and positivity with the method of in situ end-labeling of nuclear DNA fragmentation of dying cells, in the external granule cell layer of the cerebellum. Dying cells are not stained with Bcl-2 and Bax antibodies. Furthermore, no modification in the intensity of Bcl-2 and Bax protein bands and in the intensity of Bcl-2 and bax mRNA bands on Western and Northern blots, respectively, were observed between control and treated rats. These data indicate that MAM-induced apoptosis is not associated with modifications in the expression of Bcl-2 and Bax.     

Programmed cell death contributes to postnatal lung development

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.     

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.     

Conditional ablation of cerebellar astrocytes in postnatal transgenic mice

Astrocytes have been proposed to have multiple roles in the development and maintenance of the vertebrate CNS. To facilitate documentation of these roles, we designed a transgene to enable their ablation at selectable times. The transgene consists of the coding region for the herpes simplex virus-thymidine kinase (HSV-TK) under the control of the human glial fibrillary acidic protein gene promoter. The HSV-TK is innocuous but converts the antiherpetic agent ganciclovir (GCV) to a toxic product that interferes with DNA replication in proliferating cells. In a developmental study, transgenic mice were treated with GCV during the first postnatal week, with evaluation at P19. Treated mice displayed severe ataxia. Histological examination revealed disrupted astrocyte development, particularly in the cerebellum, with marked secondary effects on other cell types. Cerebellar defects included a loss in the numbers of astrocytes and an overall reduction in cerebellar size and disruption of the normally well defined cellular layers. Radial glia were disordered, Purkinje cells were ectopically distributed and displayed abnormal dendritic trees, and granule cells were markedly depleted. These effects were more severe in animals treated on postnatal day 1 versus treatment at day 5. A major factor causing granule cell death was excitotoxicity attributable to activation of NMDA receptors. These results suggest a critical role for astrocytes in cerebellar development.     

Quantitative analysis of cerebellar lobulation in normal and agranular rats

Cerebellar pattern formation was investigated in rats treated with DNA modifying agents. Animals were subjected to combinations of daily injections of methylazoxymethanol acetate (MAM) for the last 6 days gestation and/or localised X-irradiation of the hindbrain on postnatal days 1 and 5 (P1 and P5). Animals were analysed on embryonic day 18 (E18), P0, P3, P7, and P14. Five parameters of the cerebellum were recorded from midsagittal sections: the number of primary lobules; the thickness of the external germinal layer (EGL); the density of cells in the internal granule cell layer (IGL) region; and the midsagittal area and perimeter. In addition, the laterolateral cerebellar distance was calculated. The data demonstrate that pre- and postnatal reduction of the EGL results in reduced cerebellar growth and folding. Cessation of the treatment at birth results in a recovery and eventual overproduction of EGL, but cerebellar growth and the development of fissures lags behind that of normal rats. Pre- and postnatal destruction of the EGL severely limited cerebellar growth and fissuration, and the cerebella contained only five primary lobules at P14. Rats subjected to postnatal X-irradiation alone had a similar low density of granule cells relative to those treated with a combination of prenatal MAM injections and postnatal X-irradiation, and yet the cerebella contained deeper fissures and more lobules (nine at P14). The data indicate that there are two phases of cerebellar folding: the establishment of five lobules that arise independent of granule cell production, and the granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development. We propose that the lack of correlation between the severity of the granule cell loss and degree of lobulation in agranular rats indicates that granule cells exert an inductive influence over lobulation that is in part independent of the forces generated by their production and differentiation.     

Localized deposition of M-cadherin in the glomeruli of the granular layer during the postnatal development of mouse cerebellum

M-cadherin is a Ca2+-dependent cell adhesion molecule of the cadherin family, initially localized at the areas of contact between myotubes during myogenesis, but also detected in the peripheral nerve and at the adult neuromuscular junction. In this study, searching for the expression of M-cadherin in the adult mouse brain, we observed a restricted expression of M-cadherin in one of the three layers of the cerebellar cortex: the granular layer. M-cadherin was accumulated in structures rich in synapses and other intercellular junctions where mossy fibers connect granule cell dendrites, the glomeruli. This molecule was not expressed in the cerebellum during the first steps of postnatal cerebellar neurogenesis: granule cell proliferation and migration and Purkinje cell alignment. M-cadherin expression was first detected at postnatal day (P) 11, after the establishment of the synaptic connections between mossy fibers and granule cell dendrites. It then accumulated in glomeruli during their phase of maturation which is characterized by the formation of puncta adherentia between granule cell dendrites. M-cadherin was undetectable in the cerebella of the weaver and staggerer mutants, lacking granule cells, and therefore mature glomeruli and puncta adherentia. Furthermore, other components classically associated with intercellular junctions, i.e., alpha-caterin, beta-catenin and actin filaments, closely paralleled M-cadherin appearance and colocalized with M-cadherin in the mature glomeruli. M-cadherin, which appears as a molecular marker of glomerulus maturation, might be implicated in the formation, and be the ligand, of adherens junctions encountered in this structure.     

An increased number of follicles containing activated CD69+ helper T cells and proliferating CD71+ B cells are found in H. pylori-infected gastric mucosa

Multiple sclerosis is characterized by myelin destruction and oligodendrocyte loss. The neuropathological hallmark of the disease is the presence of demyelinated plaques in the central nervous system. We have recently found a gliotoxic factor in MS cerebrospinal fluid which induces programmed cell death in vitro, in glial cells. Here we show DNA fragmentation and glial cell death in biopsy samples, obtained from a patient who underwent surgery with suspicion of tumor, and whose disease record, including brain autopsy, demonstrated an active multiple sclerosis. We used the in situ TUNEL technique, a method which sensitively detects the DNA fragmentation accompanying programmed cell death in tissue sections, and compatible with classical fixation techniques. We found intense DNA fragmentation in nuclei of glial cells at-or very near-to the site of demyelination. A double labeling technique showed that glial fibrillary associated protein positive astrocytes may undergo programmed cell death in multiple sclerosis.     

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Lithium protects rat cerebellar granule cells against apoptosis induced by anticonvulsants, phenytoin and carbamazepine

We have studied the neuroprotective actions of lithium against various insults in cultured cerebellar granule cells of rats. The anticonvulsants, phenytoin and carbamazepine, have been shown to induce apoptosis of cerebellar granule cells at high concentrations. Here we found that co-presence of LiCl (1-10 mM) dose-dependently protected against phenytoin (20 microM)- and carbamazepine (100 microM)-induced neuronal apoptosis as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide metabolism, morphological inspection, chromatin condensation and DNA fragmentation. These neuroprotective effects were not prevented by inclusion of myoinositol nor mimicked by a potent inositol monophosphatase inhibitor, suggestive of a mechanism independent of inositol monophosphatase blockade. Lithium also significantly protected against apoptosis of cerebellar granule cells induced by aging of the cultures. Additionally, lithium suppressed death of cerebellar granule cells exposed to a low concentration of extracellular potassium. In contrast, it had no protective effect on cell death induced by Ca++ ionophores, a Na+ channel opener, a protein kinase inhibitor, a nitric oxide donor or H2O2. Thus, lithium has robust neuroprotective effects against apoptotic cell death induced by multiple insults with limited selectivity. These actions provide a new avenue to study the molecular and cellular mechanisms of this drug.     

Ornithine decarboxylase activity during development of cerebellar granule neurons

Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats

We determined whether fragmentation of genomic DNA, one of the hallmarks of apoptosis, occurs during structural luteolysis in cycling rats. Corpora lutea (CL) were collected from rats at each estrous cycle stage (1800 h), and fragmented DNA was extracted. Only CL from rats at the proestrous stage showed distinct DNA fragmentation. To determine the period of occurrence of DNA fragmentation, CL were collected at several points between 1200 h on the day of proestrus and 0600 h on the day of estrus. Distinct DNA fragmentation was observed from 1800 h (proestrus) to 2400 h (proestrus), and the extent was significantly lower at 0600 h (estrus). It is known that prolactin (PRL) induces structural luteolysis in rats. To examine the role of PRL in luteal DNA fragmentation, 2-bromo-alpha-ergocryptine (BE) was used to suppress the PRL surge on the day of proestrus. CL collected at 1800 h from BE-treated rats did not show distinct DNA fragmentation, and PRL injection offset the effect of BE. Histochemical analysis with a 3'-end labeling technique confirmed the occurrence of DNA fragmentation in luteal tissue. These results suggest that apoptotic cell death occurs during PRL-induced structural luteolysis.     

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.     

Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia

Delayed neuronal death after transient cerebral ischemia may be mediated, in part, by the induction of apoptosis-regulatory gene products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal cultures, and in other cell types under many different conditions. To determine whether caspase-3 serves to regulate neuronal death after cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain caspase-3; (2) examined caspase-3 mRNA and protein expression in the brain using in situ hybridization, Northern and Western blot analyses, and double-labeled immunohistochemistry; (3) determined caspase-3-like activity in brain cell extracts; and (4) studied the effect of caspase-3 inhibition on cell survival and DNA fragmentation in the hippocampus in a rat model of transient global ischemia. At 8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the hippocampus and caudate-putamen (CPu), accompanied by increased caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA and protein were predominantly increased in degenerating CA1 pyramidal neurons. Proteolytic activation of the caspase-3 precursor was detected in hippocampus and CPu but not in cortex at 4-72 hr after ischemia. Double-label experiments detected DNA fragmentation in the majority of CA1 neurons and selective CPu neurons that overexpressed caspase-3. Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor, decreased caspase-3 activity in the hippocampus and significantly reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

Susceptibility to cell death induced by mutant SV40 T-antigen correlates with Purkinje neuron functional development

Purkinje cells are uniquely susceptible to a number of physical, chemical, and genetic insults both during development and in the mature state. We have previously shown that when the postmitotic state of murine Purkinje cells is altered by inactivation of the retinoblastoma tumor susceptibility protein (pRb), immature as well as mature Purkinje cells undergo apoptosis. DNA synthesis and neuronal loss are induced in postmitotic Purkinje cells dependent upon the pRb-binding portion of SV40 large T antigen (T-ag). In the present study, Purkinje cell targeting of a mutant T-ag, PVU, which does not bind pRb, reveals disparate cerebellar phenotypes dependent upon temporal differences in transgene expression. Strong embryonic and postnatal transgene expression in three lines alters Purkinje cell development and function during the second postnatal week, causing ataxia without Purkinje cell loss. In contrast, two other transgenic lines reveal that PVU T-ag expression following normal Purkinje cell maturation causes rapid Purkinje cell degeneration. The second and third postnatal weeks of cerebellar development, which include the major period of synaptogenesis, appear to be the defining stage for the two PVU-induced phenotypes. These data indicate that Purkinje cell death susceptibility varies with developmental stage.     

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.