Single-cell analysis of mtDNA deletion levels in sporadic amyotrophic lateral sclerosis

One possible cause for the neuronal loss in sporadic amyotrophic lateral sclerosis (S-ALS) is an increase of free radicals, which may produce oxidative damage to susceptible biomolecules, which, in turn, can damage the mitochondrial DNA (mtDNA). Following laser microdissection of single motor neurons from paraffin-embedded autopsy tissue, we analyzed the presence of a common mtDNA deletion, the 5 kb common deletion (CD). Spinal cord neurons showed slightly higher CD detection rate in patients than controls (94% vs 75%). No significant differences were found between patients and controls for neurons derived from other motor or non-motor regions. A PCR assay of serial DNA dilutions (10-fold) showed no CD level differences between motor neurons in S-ALS and controls. These data suggest that neuronal death in S-ALS is not associated with significant accumulation of mtDNA deletions.     

Mitochondrial tRNA(Cys) mutation A5823G in a patient with motor neuron disease and temporal lobe epilepsy.

We discovered a new homoplasmic mutation in the mitochondrial cysteine tRNA of a 60-year-old Caucasian male suffering from asymmetrical pure lower motor neuron disease (MND) and temporal lobe epilepsy (TLE). Furthermore, titrations with Amytal, an inhibitor of NADH:CoQ oxidoreductase, revealed mild mitochondrial dysfunction in skeletal muscle tissue, which was described in patients with MND in an earlier report. The mutation was undetectable in 155 Caucasian controls of both sexes, in 40 MND patients and in 13 individuals suffering from TLE. It was, however, detected in a heteroplasmic state in the patient's mother, who did not suffer from a neurological disorder. Since this rare mutation affected a nonconserved base position and was not observed in MND or TLE materials, its relation to disease remains unclear.     

Determination of the glycogen contents in the brains of perinatal and newborn rabbits under conditions of experimental hypoxia.

The glycogen contents of 121 rabbit brains having different levels of hypoxia were determined, calculation being in mg% of glucose per g of brain. It was possible to observe significant reductions in carbohydrate content under conditions of acute and chronic hypoxia. Moderate prepartum accumulation of glycogen in untreated rabbit brains is likely. Chronic reduction in reserve fuel resulted in deficient development of the brain, as is clearly shown by brain weight determinations. This enables a parallel between this condition and intrauterine dystrophia.     

A contribution to the postnatal enzymatic state of maturity of gyrus hippocampi in the embryofetal alcohol syndrome of the rat

The influence of maternal chronic alcoholism on fetal brain development was investigated in rats. A decrease in the activity of succinate, glycerin-1-phosphate, glucose-6-phosphate dehydrogenases and of tetrazolium reductases was found. The amount of total RNA was reduced in the neonates with alcohol damage. Moreover, a retardation of myelinisation at the time of birth was observed.     

Abnormal apoptosis in chronic granulomatous disease and autoantibody production characteristic of lupus

OBJECTIVES: Patients with chronic granulomatous disease and carrier mothers of patients with chronic granulomatous disease are predisposed to developing various forms of lupus. This disorder is a neutrophil defect in intracellular killing. Abnormal apoptosis has been described. We hypothesized that abnormal apoptosis occurring in neutrophils of patients made them more immunogenic. METHODS: Human patients with chronic granulomatous disease were examined for abnormalities of neutrophil apoptosis by flow cytometry. To model the effect of abnormal apoptosis, a murine model was used. Apoptotic cells from either wild type or mice with chronic granulomatous disease were injected into either wild type or chronic granulomatous disease mice and autoantibodies were determined by ELISA. RESULTS: Our studies found that human and murine neutrophils carrying the gp91 form of chronic granulomatous disease had impaired exposure of phosphatidyl serine on the surface. Other markers of apoptosis were largely normal. Injection of apoptotic neutrophils from gp91 knockout mice into gp91 knockout mice led to the development of characteristic autoantibodies of lupus. CONCLUSIONS: Humans with chronic granulomatous disease may be at an increased risk of developing lupus due to abnormal apoptosis and abnormal clearance of apoptotic cells.     

Expression pattern of apoptotic markers in vestibular schwannomas

The Fas-Fas-L system plays a major role in the regulation of apoptosis and hence in growth in benign and malignant human tumors. As the factors regulating cell death in benign schwannomas are not well understood, we investigated the immunoexpression of the Fas-Fas-L system, as well as that of the anti-apoptotic factor Bcl-2 and the pro-apoptotic factor Bax in 14 sporadic vestibular schwannomas, and related the findings to the MIB-1 labeling index as a marker for cell proliferation. Whereas cytoplasmic Fas expression was seen in only one tumor (7%), Fas-L was found in the nuclei of 12 schwannomas (86%). Bcl-2 expression was found in the cytoplasm of 9 tumors (64%), and Bax was found in 10 out of 14 schwannomas (71%). No significant correlations between different labeling indices were observed. However, schwannomas expressing Bax tended to show a higher proliferation rate as revealed by the MIB-1 LI, suggesting a balance between cell proliferation and cell death. Our study further showed that Fas-L is present in most vestibular schwannomas; however, due to the lack of Fas expression, apoptosis in vestibular schwannomas does not seem to be mediated via the Fas-Fas-L system.     

Altered dendritic maturation and loss of GABA immunoreactivity of hippocampal neurons after prenatal exposure to chloroquine.

The aim of the study was to analyze the intrauterine and postpartal effects of Chloroquine on the dendritic maturation in the hippocampus and on the expression of GABA within hippocampal interneurons of the stratum moleculare. Fifty-nine brains of rat fetus on day 22 of gestation and 37 brains from rats from postnatal day P7 were examined. We found changes in the cytoarchitecture of hippocampal CA3 neurons on P7 day. Chloroquine treatment resulted in a significant increase of the length of the apical shafts, apical dendrites and basal dendrites of the CA3 neurons (p < 0.05) under doses comparable to serum levels reached during long-term therapy. Furthermore, an early reduction of GABA-expressing interneurons of the hippocampal striatum moleculare was observed.     

Micromolar concentrations of 2-methoxyestradiol kill glioma cells by an apoptotic mechanism,without destroying their microtubule cytoskeleton

The purpose of this study was to investigate the potential effects of 2-methoxyestradiol, a natural mammalian steroid, in glioma cells, since antiproliferative effects of this compound had been shown earlier in several leukemia and carcinoma cell lines. The effects of 0.2, 2 and 20M concentrations of 2-methoxyestradiol were measured in three malignant human glioma cell lines (U87MG, U138MG, LN405) and one malignant rat glioma cell line (RG-2) using a microtiter-tetrazolium (MTT) assay. In all cell lines, a significant reduction of the viable cell number by more then 75% occurred ( P < 0.05) for concentrations of 2 and 20M 2-methoxyestradiol after 6days. A concentration of 0.2M had smaller effects (10–40% cell reduction), which were significant in two of the cell lines tested. The apoptotic nature of cell death was further analyzed in U87MG and RG-2 cells. Caspase-3 activity was significantly induced to levels between 3.4- and 23-fold after 4days for the two higher 2-methoxyestradiol concentrations (P < 0.05). In the cell line RG-2 nuclear fragmentation was visible in many nuclei, following stains with Hoechst H33258. A round cell morphology occurred in most treated cells, which was not accompanied by a complete destruction of the microtubule network, as it can be observed with other microtubule targeting drugs. K. Chamaon: These authors contributed equally to the work.J. Stojek: These authors contributed equally to the work.