Stefan Vielhaber

Impairment of mitochondrial function in skeletal muscle of patients with amyotrophic lateral sclerosis

In skeletal muscle homogenates of 14 patients with sporadic amyotrophic lateral sclerosis, an approximately twofold lower specific activity of NADH:CoQ oxidoreductase in comparison to an age matched control group (n=28) was detected. This finding was confirmed by a detailed analysis of mitochondrial oxidative phosphorylation in skeletal muscle using saponin-permeabilized muscle fibers. (i) A significantly lowered maximal glutamate+malate and pyruvate+malate supported respiration of saponin-permeabilized fibers was detected in the patients group. (ii) Titrations with the specific inhibitor of NADH:CoQ oxidoreductase amytal revealed a higher sensitivity of respiration to this inhibitor indicating an elevated flux control coefficient of this enzyme. (iii) Applying functional imaging of mitochondria using ratios of NAD(P)H and flavoprotein autofluorescence images of saponin-permeabilized fibers we detected the presence of partially respiratory chain inhibited mitochondria on the single fiber level. A secondary defect of mitochondrial function due to the neurogenic changes in muscle seems to be unlikely since no mitochondrial abnormalities were detectable in biopsies of patients with spinal muscular atrophy. These results support the viewpoint that an impairment of mitochondria may be of pathophysiological significance in the etiology of amyotrophic lateral sclerosis.

Mitochondrial complex I deficiency in the epileptic focus of patients with temporal lobe epilepsy

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE. Ann Neurol 2000;48:766–773

Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure‐dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine‐treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH–CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high‐resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial‐encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.

The Mechanism of Neuroprotection by Topiramate in an Animal Model of Epilepsy

Summary:  Purpose: For the antiepileptic drug (AED) topiramate (TPM), neuroprotective effects have been reported in models of focal cerebral ischemia and experimental status epilepticus, but the putative mechanism of action has remained elusive.

Functional motor compensation in amyotrophic lateral sclerosis

The present study investigated the fMRI correlates of functional compensation/neural reorganization of the motor system in patients with amyotrophic lateral sclerosis (ALS). The hypothesis was that ALS patients would recruit additional brain regions compared with controls in a motor task and that activity in these regions would vary as a function of task difficulty. Patients and controls executed a motor task with two sequences (a simple and a more difficult one) of consecutive button presses. Patients and controls both activated brain regions known to be involved in motor execution and control. Activity in ipsilateral motor areas as well as difficulty–related activity in the left cerebellum could only be observed in patients. The behavioral data indicated that the motor task was much more difficult for patients than for controls. At nearly equal difficulty the observed patterns of hemodynamic activity in controls were very similar to those observed in ALS. The findings suggest that functional compensation in ALS relies on existing resources and mechanisms that are not primarily developed as a consequence of the lesion.

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.

Impaired Regulation of Brain Mitochondria by Extramitochondrial Ca2+ in Transgenic Huntington Disease Rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.

Mitochondria and Energetic Depression in Cell Pathophysiology

Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED), which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca2+ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS), mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD). However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect) is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis.

Correlation of hippocampal glucose oxidation capacity and interictal FDG-PET in temporal lobe epilepsy

Summary:  Purpose: Interictal [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) demonstrates temporal hypometabolism in the epileptogenic zone of 60–90% of patients with temporal lobe epilepsy. The pathophysiology of this finding is still unknown. Several studies failed to show a correlation between hippocampal FDG‐PET hypometabolism and neuronal cell loss. Because FDG is metabolized by hexokinase bound to the outer mitochondrial membrane, we correlated the glucose‐oxidation capacity of hippocampal subfields obtained after surgical resection with the corresponding hippocampal presurgical FDG‐PET activity.

Subfield-specific loss of hippocampal N-acetyl aspartate in temporal lobe epilepsy.

Purpose: In patients with mesial temporal lobe epilepsy (MTLE) it remains an unresolved issue whether the interictal decrease in N‐acetyl aspartate (NAA) detected by proton magnetic resonance spectroscopy (1H‐MRS) reflects the epilepsy‐associated loss of hippocampal pyramidal neurons or metabolic dysfunction.