Combined effects of resveratrol and radiation in GH3 and TtT/GF pituitary adenoma cells.

OBJECTIVE: Resveratrol and radiation decrease viability in various tumor cells. This study aims to investigate combined effects of resveratrol and radiation on viability, induction of apoptosis and necrosis, and expression of apoptosis modulators in rodent GH3 and TtT/GF pituitary adenoma cells in vitro. METHODS: Cells were incubated with 10-100 µM resveratrol. Medium and medium with ethanol served as controls. After 2 h, cells were irradiated with 0-5 Gray (Gy) and further incubated for 48-72 h. Cell viability was quantified using a hemocytometer. Cell death was assessed with an enzyme-linked immunosorbent assay (ELISA) that detects free nucleosomes in cell lysates and free nucleosomes released to the culture medium. Expression of B-cell lymphoma-2 protein (BCL-2) and BCL-2 associated Xprotein (BAX) was measured using quantitative real time-polymerase chain reaction (qRT-PCR) to analyze changes in BAX/BCL-2 ratio. RESULTS: Resveratrol and irradiation with 4 Gy alone and in combination significantly decreased cell viability (p = 0.017 and less). In the ELISA, 10 µM resveratrol significantly induced apoptosis in TtT/GF cells at 0 Gy (p < 0.001), but not at 3 or 5 Gy. In the ELISA, 10 µM resveratrol significantly induced necrosis in GH3 cells at 0, 3 and 5 Gy (p < 0.001). While qRT-PCR did not demonstrate a significant effect of 10 µM resveratrol or radiation on expression of BAX or BCL-2, a significant increase in the BAX/BCL-2 ratio was found after irradiation with 5 Gy in GH3 cells (p = 0.0027). CONCLUSION: While moderate irradiation solely led to inhibited proliferation, resveratrol induced cell death in rodent pituitary adenoma cells.

Review: iron metabolism and the role of iron in neurodegenerative disorders.

Iron plays a role for the biogenesis of two important redox-reactive prosthetic groups of enzymes, iron sulphur clusters (ISC) and heme. A part of these biosynthetic pathways takes plays in the mitochondria. While several important proteins of cellular iron uptake and storage and of mitochondrial iron metabolism are well-characterized, limited knowledge exists regarding the mitochondrial iron importers (mitoferrins). A disturbed distribution of iron, hampered Fe-dependent biosynthetic pathways and eventually oxidative stress resulting from an increased labile iron pool are suggested to play a role in several neurodegenerative diseases. Friedreich's ataxia is associated with mitochondrial iron accumulation and hampered ISC/heme biogenesis due to reduced frataxin expression, thus representing a monogenic mitochondrial disorder, which is clearly elicited solely by a disturbed iron metabolism. Less clear are the controversially discussed impacts of iron dysregulation and iron-dependent oxidative stress in the most common neurodegenerative disorders, i.e. Alzheimer's disease (AD) and Parkinson's disease (PD). Amyotrophic lateral sclerosis (ALS) may be viewed as a disease offering a better support for a direct link between iron, oxidative stress and regional neurodegeneration. Altogether, despite significant progress in molecular knowledge, the true impact of iron on the sporadic forms of AD, PD and ALS is still uncertain. Here we summarize the current knowledge of iron metabolism disturbances in neurodegenerative disorders.

The G11778A LHON mutation does not enhance ethambutol cytotoxicity in a cybrid model.

UNLABELLED: Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder, leading to a selective loss of retinal ganglion cells (RGC) and degeneration of the optic nerve, which results in severe visual impairment or even blindness. The primary causes are point mutations of the mitochondrial DNA (mtDNA), associated with aminoacid exchanges in complex I of the electron transport chain (ETC), which are thought to disturb oxidative ATP generation in the mitochondria. The major side effect of the antibiotic ethambutol, commonly used in tuberculosis therapy, is a retinopathy, which may lead to selective RGC loss, if not detected in an early stage. Moreover, LHON was reported to be elicited by ethambutol in some mutation carriers. OBJECTIVE: The present study intended to measure a possible synergism between mitochondrial dysfunction, caused by the most common LHON mutation (G11778A) and caused by ethambutol, which may lead to a higher cytotoxicity of the drug in LHON cells. MATERIAL: An NT2/D1 teratoma-derived LHON cybrid line and the parental cells. METHOD: Determination of ethambutol toxicity in both lines, using a microtiter tetrazolium assay, luminometric measurement of ATP/ADP ratios and determination of mtDNA copy numbers by Real-time PCR. RESULTS: Short-term ethambutol toxicity occurred only at micromolar concentrations, far beyond the estimated plasma peak concentrations of patients under antibiotic therapy. No significant difference occurred between both cell lines. The ATP/ADP ratios in the cybrids were surprisingly low, but showed no correlation with the mutational status of drug-treated cells. The mtDNA copy number of treated LHON and parental cells did not differ significantly. CONCLUSIONS: Ethambutol shows no synergism with the most common primary LHON mutation with respect to mitochondrial energy production or mtDNA replication in cybrid cells, although the issue of ATP decline should be further addressed in neuronally differentiated cybrids with complete OXPHOS dependency.

Absence of major accumulation of mitochondrial ND5 mutations in Parkinson patient muscle.

UNLABELLED: The pathogenesis of the selective loss of dopaminergic neurons in idiopathic Parkinson's disease (PD) has not been understood up to now. Respiratory chain dysfunction and accumulation of mitochondrial DNA deletions to biochemically relevant levels have been observed in the dopaminergic neurons. However, respiratory chain defects have also been reported in other tissues, pointing to a generalized component of oxidative stress in PD. Recently, somatic point mutations in a narrow region of the complex I polypeptide ND5 (codons 120 - 150) were suggested to separate PD patients from age-matched controls, using frontal cortex homogenates. OBJECTIVE: The present study intended to analyze whether those recently described ND5 mutations may also generally occur in skeletal muscle tissue of PD patients, in which complex I dysfunction had been measured earlier with biochemical approaches. MATERIAL: Skeletal muscle biopsy samples of 5 PD individuals with a previously characterized biochemical complex I defect and of 5 age-matched controls were used. METHOD: DNA was extracted from the muscle samples. The relevant ND5 region was PCR-cloned using a high fidelity Pfu polymerase and a low number of PCR cycles (15). Amean number of 96 clones were randomly selected from the ampicillin plates and sequenced by the dye terminator method to allow the detection of low abundance mutations with a sensitivity around 1%. RESULTS: Mutations between codons 120 and 150 were only slightly more frequent in PD versus controls (60 versus 40% of samples affected), while this ratio had been 100 versus 12.5% in frontal cortex. CONCLUSIONS: In contrast to results reported for PD frontal cortex, low-level ND5 mutations between codons 120 and 150 do not accumulate severely in biochemically affected skeletal muscle samples of PD patients.

Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases.

Inhibitor titrations were applied to characterize functional changes in mitochondrial energy metabolism in the skeletal muscle of patients with mitochondrial diseases. For this we titrated the maximal mitochondrial respiration rate of saponin-skinned muscle fibers isolated from the skeletal muscle biopsy with the specific inhibitors of mitochondrial oxidative phosphorylation complexes I, IV and V-rotenone, azide and oligomycin. For three patients with deletions of mitochondrial DNA and one patient with a complex I deficiency the titrations revealed at rather normal respiration activities of saponin-skinned fibers significant differences to healthy controls: (i) The inhibitor titration curves of the affected enzyme were much steeper and (ii) for almost complete inhibition of respiration a smaller amount of the inhibitor is necessary. The detailed analysis of the titration curves within the framework of metabolic control theory indicated elevated flux control coefficients of the respective complex of respiratory chain. On the other hand, for one patient with a mitochondrial DNA depletion syndrome, decreased respiration activities of skinned fibers but no redistribution of flux control was observed. We conclude, therefore, that application of inhibitor titrations and the quantitative description of the titration curve can be a valuable approach to elucidate functional defects of mitochondrial oxidative phosphorylation.

Laser-excited fluorescence studies of mitochondrial function in saponin-skinned skeletal muscle fibers of patients with chronic progressive external ophthalmoplegia.

The functional behavior of mitochondria in skeletal muscle of patients with chronic progressive external ophthalmoplegia was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. Variations in the mitochondrial content and the presence of partially respiratory chain-inhibited mitochondria can be detected using this novel method.

Heterogeneous tissue distribution of a mitochondrial DNA polymorphism in heteroplasmic subjects without mitochondrial disorders.

CONTEXT: Several maternally inherited point mutations of the mitochondrial genome cause mitochondrial disorders, but the correlation between genotype and phenotype remains obscure in many cases. The same mutation may cause various diseases, probably because of a different tissue distribution. OBJECTIVE: To assess the role of random somatic segregation in generating interperson differences by analysis of an apparently neutral polymorphism. DESIGN: Screening of 81 brain samples from subjects without mitochondrial disorders and selection of five necropsy cases showing a high level of heteroplasmy for the polymorphism. MAIN OUTCOME MEASURES: A proportion of various distinct genotypes in the mtDNA pool of the tissues, identified by fluorescent PCR products, representing a short polycytosine tract of variable length in the mitochondrial displacement loop. RESULTS: Differences were found between organs or groups of organs within subjects, pointing towards somatic segregation of mtDNA. In addition, marked differences of this organ distribution occurred between subjects, which cannot be explained by tissue specific selection. CONCLUSIONS: The observed interperson differences can be explained by somatic segregation, which occurs randomly at various developmental stages. Besides tissue specific selection, this process might participate in the distribution of pathogenic mtDNA mutations.

Short incubation with 2-methoxyestradiol kills malignant glioma cells independent of death receptor 5 upregulation.

We investigated the effects of 2-methoxyestradiol (2-ME), a promising new antitumor agent, on viable cell number and nuclear morphology of malignant glioma cells (three human and one rat glioma cell lines) and analyzed the controversial role of death recepor 5 (DR5) upregulation in 2-ME induced apoptosis. Microtiter-tetrazolium (MTT) assays showed a significant reduction of viable cells after incubation with 2 microM and 20 microM 2-ME for 48 and 72 hours in all cultures. In the 20 microM concentration, there were even significant effects in the majority of shorter incubation periods. Hoechst 33258 stains showed a substantial amount of cells with nuclear fragmentation indicating a late stage of apoptosis after 20 microM 2-ME treatments of 24 hours and more. The role of the DR5-mediated extrinsic apoptotic pathway was further studied in the three human glioma cell lines; 50 ng/ml of the DR5 ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and 2 microM 2-ME showed no synergism, as determined by MTT assays. Real-time PCR revealed no significantly increased amount of DR5 mRNA, suggesting that receptor upregulation does not play a major role for 2-ME-induced apoptosis in glioma cells, in contrast to data for a breast cancer cell line in the literature.

Spatial and temporal disease progression of adult-onset subacute sclerosing panencephalitis.

An adult-onset case of subacute sclerosing panencephalitis with occipitofrontal spread of the infection documented clinically and by MRI is reported. Autopsy revealed numerous intranuclear viral inclusions and widespread demyelination in both frontal lobes. In the occipital lobes where the disease started 5 years previously, inclusions were rare, but degenerative tissue changes were prominent. This case underlines the importance of measles virus migration for the progression of this fatal disorder.

Effects of phospholipase Cgamma on Polo-like kinase 1 expression in human glioma cells.

PURPOSE: The precise regulation of the mammalian PLK-1 gene, involved in progression of the G2/M transition, remains to be clarified. Phospholipase Cgamma, associated with cell proliferation and invasion in human gliomas, could be one of the important candidates for the modulation of PLK-1 expression. Therefore, we studied the correlation between PLCgamma activity and PLK-1 expression and determined cell size and proliferation after PLCgamma inhibition in two glioma cell lines and a primary culture of a glioblastoma. METHODS: The glioma cells were investigated with highly specific chemical and antisense inhibition of PLCgamma. The effects of the inhibition were checked by means of morphometrical, semiquantitative immunohistochemical methods, and MTT-assays. RESULTS: The chemical and antisense inhibition of PLCgamma resulted in decreased expression of PLK-1 and reduced cell density in glioma cell lines as well as in a primary culture of a glioblastoma. These findings were verified by MTT-assays. CONCLUSION: The activity of PLCgamma seems to modulate the expression of PLK-1. In further experiments serum-depleted cultures, stimulated by different growth factors, should be used after inhibitor pretreatment to study the in vivo conditions.

Visualization of defective mitochondrial function in skeletal muscle fibers of patients with sporadic amyotrophic lateral sclerosis.

The mitochondrial function in skeletal muscle was investigated in skeletal muscle biopsies of 26 patients with sporadic amyotrophic lateral sclerosis (ALS) and compared with investigations of 28 age-matched control muscle samples and biopsies of 6 patients with spinal muscular atrophy (SMA) and two patients with Tay-Sachs disease. In comparison to the control, SMA and Tay-Sachs biopsies, we observed in the ALS samples a significant about two-fold lower activity of complex I of mitochondrial respiratory chain. To visualise the distribution of the mitochondrial defect in skeletal muscle fibers we applied confocal laser-scanning microscopy and video fluorescence microscopy of NAD(P)H and fluorescent flavoproteins. The redox change of mitochondrial NAD(P)H and flavoproteins on addition of mitochondrial substrates, ADP, or cyanide were determined by measurement of fluorescence intensities with dual-photon UV-excitation and single-photon blue excitation. In skeletal muscle fibers of ALS patients with abnormalities of mitochondrial DNA (multiple deletions, n=1, or lower mtDNA levels, n=14) we observed a heterogeneous distribution of the mitochondrial defects among individual fibers and even within single fibers. In some patients (n=3) a mitochondrial defect was also detectable in cultivated skin fibroblasts. These findings support the viewpoint that the observed impairment of mitochondrial function in muscle of certain ALS patients is caused by an intrinsic mitochondrial defect which may be of pathophysiological significance in the etiology of this neurodegenerative disease.

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas.

The arachnoideal compartment provides the vascular sources for three different tumor types rich in vessels: angiomatous meningioma, some atypical meningioma with high vascularity and meningeal hemangiopericytoma. We investigated immunohistochemically the expression and distribution of vascular mitogenes in 7 angiomatous meningiomas, 8 atypical meningiomas with high vasculature and 4 hemangiopericytomas. On the one hand it should be studied which vascular growth factors such as VPF/VEGF-1, VPF/VEGF-2, bFGF, PDGF and TGF-alpha could be responsible for the close meshwork of vessels within the tumors. On the other hand we were interested in whether or not there are differences in vascular mitogens between slowly growing angiomatous meningiomas and both other types with their increased tendency to recur. PDGF and TGF-alpha were extensively expressed in the endothelium and smooth muscle cells of the vessels, as well as tumor cells. VEGF-2 could only be found in endothelial cells of all three tumor entities. bFGF was localized in some vessels of angiomatous meningiomas and VEGF-1 revealed a very low expression with a localization comparable with VEGF-2. Moreover, uPAR was diffusely expressed in nearly all tumor cells and endothelial cells. The fact that tumor cells of hemangiopericytomas and meningiomas did not show any immunohistochemical reaction with VEGF's could indicate a lower priority of these growth factors for neovascularization in this type of neoplasm. A different expression of vascular mitogens between benign angiomatous meningiomas and atypical meningiomas as well as hemangiopericytomas with their tendency for recurrence could not be observed. The morphological evidence for extravasates of IgG-proteins, Fibrin and Fibronectin due to VPF-effects seems not to be a renouncable condition for neoangiogenesis in the tumors investigated.

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Correlation of TGF-alpha and EGF-receptor expression with proliferative activity in human astrocytic gliomas.

Fifty-nine paraffin-embedded astrocytic gliomas (four WHO grade 1, 21 WHO grade 2, 17 WHO grade 3 and 17 glioblastomas, WHO grade 4) were immunohistochemically investigated for expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncoprotein c-erbB-2 by semiquantitative assessment. Proliferative activity was simultaneously analyzed by using the antibody Ki-67 (MIB-1). Immunostaining in neoplastic cells was quantified by image analysis. Concerning the antibodies used, the percentage of immunoreactive cells increased with histologic malignancy. There was no expression of EGF-R and c-erbB-2 in the majority of low-grade astrocytomas. However, small focal expressions of TGF-alpha and EGF-R were observed in several low-grade astrocytomas (11/25), suggesting an early stimulation of malignant transformation. With regard to percentage, a strong positive correlation between TGF-alpha and EGF-R-stained cells was found, indicating an autocrine stimulation of the mitogenic pathway of the TGF-alpha/EGF-R system. Likewise, indices of EGF-R and c-erbB-2 positive cells correlated significantly. Less significant correlations were also seen between EGF-R, c-erbB-2 frequencies and the Ki-67 labeling index. However, there was no correlation between TGF-alpha and Ki-67 indices. The results suggest that TGF-alpha expression is not directly related to the proliferative potential as judged by the Ki-67 labeling index. Furthermore, besides EGF-R and c-erbB-2, other growth factors and their receptors or mutant EGF-R might participate in the proliferative activity of gliomas.

Mdr1 mRNA expression differs between grade III astrocytomas and glioblastomas.

104 N2-frozen samples from 33 astrocytic tumors previously untreated with cytotoxic drugs have been analyzed for the expression of p-glycoprotein transcripts (mdrl) by RT-PCR using beta2-microglobulin as an internal control. A remarkable variation was observed even within a single tumor in 50% of the cases. Nevertheless, a difference became visible between the groups of anaplastic astrocytomas and glioblastomas. While 78% of the grade 3 astrocytomas contained at least a minimum of 1 sample with a very low mdr1 expression, this was the case only in 23% of the glioblastomas. This supports an earlier observation revealing a positive correlation between tumor grading and the tumor cell fraction stained with the monoclonal antibody JSB1. On the other hand, no major differences were found between the histological groups when the samples with the highest mdr1 expression were selected to represent the individual tumors. Those samples are less informative. They might be derived from tumor regions in which capillaries deliver a larger fraction of the total mRNA pool. No induction of mdr1 was observed in some early astrocytoma or glioblastoma cell cultures even after administration of high concentrations of the drugs ACNU and VM26, often used in glioma therapy.

Stiff-man syndrome: possible autoimmune etiology targeted against GABA-ergic cells.

We report the case of a female patient, who died at the age of 66 years. Besides an insulin-dependent diabetes mellitus (IDDM) she had developed the clinical symptoms of stiff-man-syndrome (SMS) and harbored autoantibodies against glutamate-decarboxylase (GAD) in blood and liquor. GAD catalyzes the biosynthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The autopsy revealed typical alterations observed in diabetes mellitus including an incomplete fibrosis of pancreatic Langerhans islets. A decrease of GABA-ergic cells in the cerebellar cortex was observed, and a size reduction of Renshaw cells in the spinal cord. Furthermore, a dilution series of a polyclonal GABA antibody delivered a reduced immunofluorescence in the cerebellum. In skeletal muscle a neurogenic atrophy was observed. As described in literature, the clinical symptoms decayed following clonazepam administration. We suggest that this case including GAD autoantibodies, dramatic loss of GAD-expressing pancreatic cells, and loss or atrophy of GABA secretory neurons, supports the hypothesis that SMS may be an autoimmune disease directed against GABA-ergic cells. Furthermore, we suggest a neuronal hypersensitivity at the spinal cord level caused by the atrophic Renshaw cells.

MGMT- and P450 3A-inhibitors do not sensitize glioblastoma cell cultures against nitrosoureas.

UNLABELLED: AIM, MATERIAL AND METHODS: Using RT-PCR and immunohistochemistry the expression of cytochrome P450 was evaluated in a series of 22 glioblastomas and 4 anaplastic astrocytomas (WHO grade III). Since rat liver P450 can catalyze the denitrosation of the nitrosourea compound BCNU in vitro, cell culture experiments were performed to test a possible sensitizing effect of P450 3A inhibitors (tiamulin and ketoconazole) in BCNU treatment of glial tumor cells. O6-benzylguanine (BG), an inhibitor of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT), was used in parallel experiments, since MGMT is discussed as a main mechanism in nitrosourea resistance. RESULTS: RT-PCR reactions with primers designed according to the sequences for CYP1A1 and CYP2E1 were always positive, while those for CYP1A2 and CYP2D6 were negative. The strongest PCR products were detected with CYP3A primers, but CYP3A expression was heterogeneous within the tumor samples. Antibodies to human liver CYP3A4 stained a subfraction of tumor cells (18% of the cells in glioblastomas and 14% in grade III astrocytomas) and to some extend neurons in normal brain areas, while astrocytes were negative. For cell culture experiments with P450 3A and MGMT inhibitors, early passages of 3 glioblastomas, a late passage of an immortalized cell line derived from a reoccurring glioblastoma, and the human glioblastoma line LN405 were used. The sensitivity of the tumor cells for both nitrosourea compounds was very low, when low concentrations were applied (comparable to the achievable blood concentrations in glioma patients). Strong effects did only occur when the concentrations were raised 9-fold or 27-fold. CONCLUSION: In no case could a significant sensitizing effect of P450 3A- and MGMT inhibitors be demonstrated.

Expression of matrix metalloproteinases in a series of 12 meningiomas.

The expression of metalloproteinases was evaluated in a series of 12 meningiomas of various histological subtypes including 3 meningotheliomatous, 3 fibroblastic, 4 transitional and one psammomatous meningioma (WHO grade I) as well as one anaplastic meningioma (WHO grade III). No gelatinolytic activity could be detected in all tumor samples pointing towards no or very low activity of both MMP-2 and MMP-9. At least MMP-2 mRNA could be found in 10 out of 12 tumor samples by the reverse transcription PCR method (RT-PCR) followed by electrophoresis on silver-stained polyacrylamide gels, which allows the detection even of small traces of a specific mRNA. The PCR products were identified as MMP-2 sequences without introns (mRNA-derived) by direct sequencing, thereby demonstrating a low transcriptional activity of the gene. The translation of these mRNAs, however, did not result in amounts of protein detectable by immunohistochemistry or Western blotting. Therefore, neither MMP-2 nor MMP-9 should play a major role for tumor growth within dura mater or bone structures or for brain infiltration in our tumor series. Therefore, other mechanisms must be responsible for extracellular matrix degradation at least in a fraction of meningiomas.

Dysregulation of iron protein expression in the G93A model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons which leads to progressive paralysis and death by respiratory failure. Although the cause of sporadic ALS is still unknown, oxidative stress is suggested to play a major role in the pathogenesis of this disease and of the rare familial form, which often exhibits mutations of the superoxide dismutase 1 (SOD1) gene. Since enhanced iron levels are discussed to participate in oxidative stress and neuronal death, we analyzed the expression levels of Fe-related mRNAs in a cell culture ALS model with the G93A mutation of SOD1. We observed an increased total iron content in G93A-SOD1 SH-SY5Y neuroblastoma cells compared to wild-type (WT)-SOD1 cells. mRNA expression for transferrin receptor 1 (TfR1) and divalent metal transporter 1 was increased in G93A-SOD1 cells, which was in accordance with higher iron uptake. Experiments with the iron chelator deferoxamine revealed a normal reaction of WT and mutant cells to cytoplasmic iron depletion, i.e. TfR1 upregulation, suggesting a basically conserved function of the iron-responsive element/iron regulatory protein (IRE/IRP) pathway, designed to adapt gene expression to iron levels. Expression levels of mitoferrin 1 and 2, frataxin, and iron-sulfur cluster scaffold protein were also significantly increased in G93A-SOD1 cells, suggesting higher mitochondrial iron import and utilization in biosynthetic pathways within the mitochondria. Moreover, expression of these transcripts was further enhanced, if G93A-SOD1 cells were differentiated by retinoic acid (RA). Since RA treatment increased cytoplasmic reactive oxygen species (ROS) levels in these cells, an IRE/IRP independent, ROS-mediated mechanism may account for dysregulation of iron-related genes.