Marco Sparaco

Mitochondrial dysfunction and migraine: evidence and hypotheses

The molecular basis of migraine is still not completely understood. An impairment of mitochondrial oxidative metabolism might play a role in the pathophysiology of this disease, by influencing neuronal information processing. Biochemical assays of platelets and muscle biopsies performed in migraine sufferers have shown a decreased activity of the respiratory chain enzymes. Studies with phosphorus magnetic resonance spectroscopy (31P-MRS) have demonstrated an impairment of the brain oxidative energy metabolism both during and between migraine attacks. However, molecular genetic studies have not detected specific mitochondrial DNA (mtDNA) mutations in patients with migraine, although other studies suggest that particular genetic markers (i.e. neutral polymorphisms or secondary mtDNA mutations) might be present in some migraine sufferers. Further studies are still needed to clarify if migraine is associated with unidentified mutations on the mtDNA or on nuclear genes that code mitochondrial proteins. In this paper, we review morphological, biochemical, imaging and genetic studies which bear on the hypothesis that migraine may be related to mitochondrial dysfunction at least in some individuals.

Friedreich's ataxia: Oxidative stress and cytoskeletal abnormalities

Friedreichs ataxia (FRDA) is an autosomal recessive disorder caused by mutations in the gene encoding frataxin, a mitochondrial protein implicated in iron metabolism. Current evidence suggests that loss of frataxin causes iron overload in tissues, and increase in free-radical production leading to oxidation and inactivation of mitochondrial respiratory chain enzymes, particularly Complexes I, II, III and aconitase. Glutathione plays an important role in the detoxification of ROS in the Central Nervous System (CNS), where it also provides regulation of protein function by glutathionylation. The cytoskeletal proteins are particularly susceptible to oxidation and appear constitutively glutathionylated in the human CNS. Previously, we showed loss of cytoskeletal organization in fibroblasts of patients with FRDA found to be associated with increased levels of glutathione bound to cytoskeletal proteins. In this study, we analysed the glutathionylation of proteins in the spinal cord of patients with FRDA and the distribution of tubulin and neurofilaments in the same area. We found, for the first time, a significant rise of the dynamic pool of tubulin as well as abnormal distribution of the phosphorylated forms of human neurofilaments in FRDA motor neurons. In the same cells, the cytoskeletal abnormalities co-localized with an increase in protein glutathionylation and the mitochondrial proteins were normally expressed by immunocytochemistry. Our results suggest that in FRDA oxidative stress causes abnormally increased protein glutathionylation leading to prominent abnormalities of the neuronal cytoskeleton.

New Morphological Approaches to the Study of Mitochondrial Encephalomyopathies

Molecular genetics, biochemistry, immunology and morphology, are being applied in a coordinated fashion to unveil the molecular basis of the mitochondrial encephalomyopathies. Mutations of mitochondrial DNA (mtDNA) have been found in well characterized clinical groups of these disorders. New and old morphologic methods have been applied to investigate muscle biopsies from patients with mtDNA mutations. Important observations have been made on the cellular localization of normal and mutated mtDNA and on the expression of mtDNA‐encoded polypeptides. These observations have provided insight into the pathogenesis of respiratory chain enzyme deficiency at the level of individual muscle fibers. Application of immunocytochemical and in situ hybridization techniques at the electron microscopic level will extend these studies to the level of individual mitochondria.

Protein glutathionylation in human central nervous system: Potential role in redox regulation of neuronal defense against free radicals

Neuronal defense against free radicals is mediated primarily by the glutathione system. A cerebral defect of this system gives rise to the oxidative stress occurring in some neurological diseases. Glutathione provides a means of regulating protein function by glutathionylation, consisting of the formation of mixed disulfides between cysteines and glutathione. The glutathionylation of proteins, during both constitutive metabolism and oxidative stress, represents for the cell a mechanism to link physiological processes, and/or adaptive stress responses, to changes in intracellular redox states. In this study, we analyzed the topographic distribution of the protein glutathionylation normally occurring in human central nervous system. Constitutively glutathionylated proteins appeared uniformly distributed throughout all cortical layers of the cerebral and cerebellar cortex as well as throughout the gray matter of the spinal cord. The degree of immunocytochemical staining was clear in neurons, mild in oligodendrocytes, and weaker in astrocytes. The proteins preferentially glutathionylated were cytoskeletal proteins. Our results suggest a potential role of glutathionylation in the redox regulation of neuronal survival and in the control of axon/dendrite stability.

Myoclonic Epilepsy with Ragged‐red Fibers (MERRF): An Immunohistochemical Study of the Brain

Myoclonic epilepsy with ragged‐red fibers (MERRF) is a maternally inherited disorder of oxidative phos‐phorylation due to specific point mutations within the mitochondrial tRNALvs gene. Mitochondrial dysfunction in the central nervous system (CNS) of patients with MERRF accounts for the neurological manifestations of the disease. Antibodies against subunits of complex I, III, IV and V of the respiratory chain were used to study the expression of these proteins in the frontal cortex, cerebellum and medulla from an autoptic case of MERRF. We found a selective decreased expression of subunit II of cytochrome c oxidase (COX‐II) in these regions. Immunohistochemical abnormalities were more widespread than the lesions described by traditional histopathological techniques and made possible an attempt of explanation for the neurological symptoms of the patient.

Focal cytochrome c oxidase deficiency in the brain and dorsal root ganglia in a case with mitochondrial encephalomyopathy (tRNAIle 4269 mutation): histochemical, immunohistochemical, and ultrastructural study

This is the first report with histochemical and immunohistochemical techniques of an autopsy case with mitochondrial encephalomyopathy caused by the mitochondrial tRNA(Ile) (nt4269) A to G mutation showing focal cytochrome c oxidase (COX) deficiency of neuronal cells. The 18-year-old male patient had cardiomyopathy, hearing disability, mental retardation, and seizures. Muscle biopsy exhibited many ragged-red fibers and focal COX deficiency. A postmortem histochemical study on frozen sections of the cerebral cortex, cerebellum, brain stem, and dorsal root ganglia revealed a loss of COX activity in some neuronal cells. On immunohistochemical staining, COX was also defective in a mosaic pattern. Focal COX deficiency may cause variable neurological manifestations in mitochondrial encephalomyopathies.

Cerebral Venous Thrombosis and Headache – A Case‐Series

Headache happens in the majority of patients with Cerebral Venous Thrombosis (CVT) being sometimes the sole manifestation of the disease. Herein we report a case‐series of CVT, focusing on headache characteristics.

Frataxin Silencing Inactivates Mitochondrial Complex I in NSC34 Motoneuronal Cells and Alters Glutathione Homeostasis

Friedreich’s ataxia (FRDA) is a hereditary neurodegenerative disease characterized by a reduced synthesis of the mitochondrial iron chaperon protein frataxin as a result of a large GAA triplet-repeat expansion within the first intron of the frataxin gene. Despite neurodegeneration being the prominent feature of this pathology involving both the central and the peripheral nervous system, information on the impact of frataxin deficiency in neurons is scant. Here, we describe a neuronal model displaying some major biochemical and morphological features of FRDA. By silencing the mouse NSC34 motor neurons for the frataxin gene with shRNA lentiviral vectors, we generated two cell lines with 40% and 70% residual amounts of frataxin, respectively. Frataxin-deficient cells showed a specific inhibition of mitochondrial Complex I (CI) activity already at 70% residual frataxin levels, whereas the glutathione imbalance progressively increased after silencing. These biochemical defects were associated with the inhibition of cell proliferation and morphological changes at the axonal compartment, both depending on the frataxin amount. Interestingly, at 70% residual frataxin levels, the in vivo treatment with the reduced glutathione revealed a partial rescue of cell proliferation. Thus, NSC34 frataxin silenced cells could be a suitable model to study the effect of frataxin deficiency in neurons and highlight glutathione as a potential beneficial therapeutic target for FRDA.

Immunolocalization of heat shock proteins in ragged-red fibers of patients with mitochondrial encephalomyopathies

Monoclonal antibodies against the 60 kDa heat shock protein (HSP-60) and against ubiquitin (UB) were used to study the expression of these proteins in muscle samples from patients with qualitative and quantitative alterations of mitochondrial DNA (mtDNA). We found an enhanced expression of HSP-60 and UB that was preferentially localized in ragged-red fibers (RRFs). HSP-60 may act as a protein repair enzyme catalyzing the refolding of misfolded proteins in the matrix of mitochondria of RRFs. On the other hand, UB could promote the elimination of abnormal proteins by its covalent interaction to substrates.