Hypoxia increases activity of the BK-channel in the inner mitochondrial membrane and reduces activity of the permeability transition pore.

Hypoxia can cause severe damage to cells by initiating signaling cascades that lead to cell death. A cellular oxygen sensor, other than the respiratory chain, might exist in sensitive components of these signaling cascades. Recently, we found evidence that mitochondrial ion channels are sensitive to low levels of oxygen. We therefore studied the effects of hypoxia on the mitochondrial BK-channel (mtBK), on the mitochondrial permeability transition pore (PTP), and on their possible interaction. Using single-channel patch-clamp techniques we found that hypoxia inhibited the PTP but substantially increased the mtBK activity of mitoplasts from rat liver and astrocytes. Experiments measuring the mitochondrial membrane potential of intact rat brain mitochondria (using the fluorescence dye safranine O) during hypoxia exhibited an increased Ca(2+)-retention capacity implying an impaired opening of the PTP. We also found a reduced Ca(2+)-retention capacity with 100 nM iberiotoxin, a selective inhibitor of BK-channels. We therefore conclude that there is interaction between the mtBK and the PTP in a way that an open mtBK keeps the PTP closed. Thus, the response of mitochondrial ion channels to hypoxia could be interpreted as anti-apoptotic.

Unusual presentations of patients with the mitochondrial MERRF mutation A8344G.

MERRF is typically characterized by myoclonus, generalized seizures and ragged-red fibers in muscular biopsy. We report a family (harbouring the A8344G mutation) with a late onset of the disease and an uncommon clinical manifestation, including episodes of reversible respiratory failure, the presence of ophthalmoplegia, and the absence of seizures and myoclonus in most subjects. We conducted histochemical, biochemical and molecular genetic studies. Mutation analysis revealed that the level of mutated mitochondrial DNA (mtDNA) was above 80% in the skeletal muscle of all siblings. Nevertheless, one severely affected individual did neither present cytochrome c oxidase-negative fibers nor ragged-red fibers in the skeletal muscle biopsy. These data extend the phenotypic range associated with the MERRF syndrome. We suggest that the analysis of mtDNA could be of importance in many cases of unclear multisystem disorders in later life.

The neurotrophin receptor TrkB is colocalized to mitochondrial membranes.

The transmembrane tyrosine-specific protein kinase TrkB has been shown to serve as a receptor for the neurotrophic factors BDNF and NT-4. Neurotrophin binding to TrkB isoformes mediates many intracellular signaling pathways, including calcium signalling. Two truncated isoforms of the receptor, lacking the tyrosine kinase activity, signal through a yet unknown pathway. Specific signals modulate the surface expression of TrkB, which is localized in considerable amounts in intracellular pools. These intracellular pools has not been specified so far. We therefore investigated the intracellular distribution of TrkB by colocalisation studies. In contrast to the unspecific neurotrophin receptor NGFRp75, TrkB immunohistochemistry showed a staining pattern very similar to mitochondrial stainings in adult human skeletal muscle fibers. Immunofluorescence techniques revealed in different types of permeabilized cells that TrkB is bound to mitochondrial membranes. This observation was confirmed on isolated astrocyte mitoplasts. Colocalisation of the TrkB ligand NT-4 and the specific mitochondrial marker cytochrome c oxidase was also observed. Western blot analysis of isolated mitochondria from rat brain and skeletal muscle verified that a truncated isoform of TrkB is present in both, brain and muscle mitochondria, and full-length TrkB is additionally present in brain mitochondria. Our results imply that neurotrophins can be stored in mitochondria and possibly act as signalling molecules on mitochondria.

Analysis of a single nucleotide polymorphism in codon 388 of the FGFR4 gene in malignant gliomas.

The FGFR4 codon 388 polymorphism (Arg(388), Arg/Gly(388) or Gly(388)) was determined in glioblastoma multiforme (GBM), anaplastic astrocytomas (AA), diffuse astrocytomas (DA), and control muscles. Arg(388) was rare in AA, GBM, muscles, and was absent in DA. The Arg/Gly(388) and the Gly(388) frequency was equal among GBM and controls. FGFR4 expression was not related to codon 388 in GBM, and no survival differences between Arg/Gly(388) and Gly(388) tumors were found. U87 cells (Arg/Gly(388)) did not show higher invasion than U138 cells (Gly(388)). This suggests that the FGFR4 codon 388 status does not play a major role in malignant gliomas.

Assessment of tumor cell invasion factors in gliomatosis cerebri.

Gliomatosis cerebri (GC) is a rare brain tumor characterized by widespread infiltration of large parts of the brain and sometimes even the spinal cord. To determine the cause of this extraordinary degree of brain invasion, we studied immunoexpression of factors associated with brain infiltration in low-grade and high-grade tumor samples from nine GC cases. We further determined the allelic status of the fibroblastic growth factor receptor 4 (FGFR4) gene at position 388 (arginine [Arg(388)] or glycine [Gly(388)]) in eighteen GC patients, because the presence of at least one Arg(388) allele has been suggested to favor tumor cell motility compared to tumor cells homozygeous for the Gly(388) allele. Immunohistochemical analyses showed that tumor samples from three GC cases expressed Tenascin-C, whereas six cases had CD44 - immunopositive tumor samples. Expression of MMP-9 was not observed in any of the nine GC patients. FGFR4 genotyping revealed the presence of the Arg(388) in 72% of the eighteen GC cases, a frequency similar to the one found in 21 common astrocytomas (71%). In tumor-free control DNA, the Arg(388) phenotype was present in 60%. These data indicate that CD44 expression might be related to the tumor infiltration in GC, and that patients suffering from GC or other common astrocytomas do not have a significantly increased frequency of the tumor cell motility-favoring Arg(388) FGFR4 allele.

Effect of coenzyme Q10 on the mitochondrial function of skin fibroblasts from Parkinson patients.

Several lines of evidence suggest an impairment of mitochondrial function in the brain of patients with Parkinson's disease (PD). However, the presence of a detectable mitochondrial defect in extracerebral tissue of these patients remains a matter of dispute. Therefore, we investigated mitochondrial function in fibroblasts of 18 PD patients applying biochemical micromethods. Putative beneficial effects of coenzyme Q(10) (CoQ(10)), a potent antioxidant, on the mitochondrial function of skin fibroblast cultures were evaluated. Applying inhibitor titrations of the mitochondrial respiration to calculate flux control coefficients of respiratory chain complexes I and IV, we observed deficiencies of both complexes in cultivated skin fibroblasts of PD patients. Cultivation of fibroblasts in the presence of 5 microM CoQ(10) restored the activity of impaired respiratory chain complexes in the fibroblast cultures of 9 out of 18 PD patients. Our data support the presence of a generalised mitochondrial defect in at least a subgroup of patients with PD that can be partially ameliorated in vitro by coenzyme Q(10) treatment.

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 DNA and respiratory chain function in spinal cords of ALS patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron death. In order to address the question of a putative role of mitochondrial dysfunction in the pathogenesis of ALS, we studied the mitochondrial DNA (mtDNA) and mitochondrial respiratory chain enzyme activities in spinal cords of ALS patients and in control subjects without neuropathologic abnormalities. Using a "double PCR and digestion" technique to estimate the levels of randomly distributed point mutations in two small regions of the mtDNA, we found significantly higher levels of mutant mtDNA in the spinal cord of ALS patients compared to controls. No large-scale rearrangements were found, but the amount of mtDNA, measured by Southern blot, was significantly lower in the ALS samples. This reduction correlated well with a decrease of citrate synthase (CS) activity, a mitochondrial marker, as were the activities of respiratory chain complexes I + III, II + III, and IV, suggesting a loss of mitochondria in ALS spinal cords.