Mitochondrial encephalomyopathies

Over the past two decades, there have been increasing reports of human disorders due to mitochondrial respiratory chain dysfunction. The mitochondria are the ATP-generating organelles in mammalian cells and contain their own DNA (mtDNA) which is maternally inherited. ATP is produced via oxidative phosphorilation through 5 respiratory complexes, whose subunits are encoded by both mtDNA and nuclear DNA (nDNA) genes. This dual genetic control explains why mitochondrial disorders can be transmitted by either mendelian or maternal genetics. Both normal and mutated mtDNA may coexist within patient’s tissues (heteroplasmy). The phenotypic expression of a mutation depends on the amount of mutated mtDNA in each tissue, and on the tissue-specific threshold level. MtDNA haplogroups may further confer a genetic susceptibility basis for various disorders. Due to their higher energy demands, skeletal muscle, heart and brain are more severely involved. Nevertheless, given the essential role of oxidative phosphorilation for tissue function, virtually all tissues and organs can be involved.

Once a mitochondrial disease is suspected on clinical and laboratory (mainly muscle biopsy features) grounds, definite diagnosis only comes from the detection of specific molecular defects, which allows definition of inheritance pattern and genetic counseling. Several mtDNA mutations have been associated with specific clinical patterns, although the degree of variability out-weights what expected from a strict genotype-phenotype correlation, indicating that other factors, including those derived from mt and nDNA background, are likely to involved. Furthermore many patients with clinically and morphologically defined mitochondrial disorders belong to none of the known genetic categories. The role of the nuclear genes in the mitochondrial disorder aetiopathogenesis has yet to be completely defined. It is however clear that different steps of mitochondrial biogenesis and function appear to be involved. For instance, disorders with early lethality are associated with recessive mutations affecting genes that belong to the biosynthetic pathway or to assembling factors of Cytochrome c Oxidase (such as SCO!, SCO2, COX10 and SURF1). On the other end, adult-onset Progressive External Ophthalmoplegias are due either to autosomal dominant mutations of ANT1, Twinkle and POLG1 or to recessive mutations of POLG1, all of them resulting in an unstable mtDNA. Other enzymatic activities involved in the mtDNA metabolism may cause infantile disorders with mtDNA depletion. Unraveling the pathogenesis of these mitochondrial disorders may offer therapeutic tools to treat often devastating disorders.

Metabolic Myopathies

This field of muscle disorders has been present from the onset of the Dino Ferrari Center activity. It includes the study of defects of the glycogen, glucose and lipid metabolism. Recently we characterized some of these disorders.

Deficiency of amylo-1,6-glucosidase, 4-alpha-glucanotransferase enzyme (AGL or glycogen debrancher enzyme) is responsible for glycogen storage disease type III, a rare autosomal recessive disorder of glycogen metabolism. The AGL gene is located on chromosome 1p21, and contains 35 exons translated in a monomeric protein product. The disease has recognized clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different subjects. The clinical manifestations of GSD III are represented by hepatomegaly, hypoglycemia, hyperlipidemia, short stature and, in a number of subjects, cardiomyopathy and myopathy. The disorder presents a large genotypic-phenotypic heterogeneity of GSD III, thus preventing a strategy of mutation finding based on screening of recurrent common mutations (Lucchiari et al. 2001).

We also describe a new metabolic myopathy, muscle enolase deficiency, in a 47-year-old man affected with exercise intolerance and myalgias (Comi et al. 2001). The enzyme enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. In adult human muscle, over 90% of enolase activity is accounted for by the beta-enolase subunit, the protein product of the ENO3 gene. In the described patient, the beta-enolase protein was dramatically reduced in the muscle of our patient, by both immunohistochemistry and immunoblotting, while alpha-enolase was normally represented. The ENO3 gene of our patient carries two heterozygous missense mutations affecting highly conserved amino acid residues; a G467A transition changing a glycine residue at position 156 to aspartate, in close proximity to the catalytic site, and a G1121A transition changing a glycine to glutamate at position 374. These mutations were probably inherited as autosomal recessive traits since the mother was heterozygous for the G467A and a sister was heterozygous for the G1121A transition. Our data suggest that ENO3 mutations result in decreased stability of mutant beta-enolase. Muscle beta-enolase deficiency should be considered in the differential diagnosis of metabolic myopathies due to inherited defects of distal glycolysis.

Ageing and neurodegenerative disorders

Mitochondrial DNA mutations affecting the regions controlling mtDNA replication and transcription increase with age, therefore suggesting a role in the ageing process and in the ageing-related neurodegenerative disorders (Michikawa et al 1999). The accumulation of mutations in the D-loop and adjacent transcription promoters correlates with the histochemical cytochrome c oxidase phenotype in the aged muscle (Del Bo et al., 2003).

Similar data are observed in fibroblasts obtained from relatively young Down Syndrome (DS) subjects, therefore suggesting that mtDNA involvement may contribute to ageing-associated phenomena in DS, such as the Alzheimer-like cognitive decline (Del Bo et al. 2001). A direct role of mitochondrial dysfunction in the pathogenesis of neurodegenerative disorder is ssupported by the findingof a specific mtDNA mutation in the Subunit I of Complex IV in a patient with motor neuron disease (Comi et al, 1998).

The Down Syndrome is a potential model for some of the neuropathologic changes of Alzheimer Disease. Multifactorial agents that may modulate the clinical phenotype may therefore be evaluated in this condition. This approach led us to establish an additive effect of the apoliprotein e4 haplotype and the Met 129 allele of the Prion protein on the rate of cognitive decline of these subjects (Del Bo et al.1997; Del Bo et al. 2003).

Cellular Mediated Gene Therapy

Stem cell transplantation is a potential therapeutic strategy for the treatment of neurodegenerative diseases and muscular dystrophies. Recent evidence suggests that somatic stem cells may differentiate into tissues different from those where they reside. The extent and degree of transdifferentiation of adult somatic stem cells are a controversial issue. Cell fusion rather than true phenotype change can account for this phenomenon.

• In vitro myogenic differentiation of Bone Marrow cells
  We investigated the myogenic potential of mouse Bone Marrow (BM) cells evaluating the expression of skeletal muscle markers and the generation of myotubes.
  We demonstrated the expression of striated muscle specific markers by BM cells after isolation and in muscle medium culture. We observed the presence of both markers of early myogenic program such as PAX3, Myf5, MyoD, desmin and late myogenesis such as myosin heavy chain and a-sarcomeric actin. These markers are detected by immunocytochemistry, Western blot and RT-PCR. We generated BM derived clones that are able to fully differentiate in myotubes.
  
• BM cells contribution to muscle repair in the mdx Dystrophic Mouse
  To investigate whether the transplantation of BM myogenic cells into mdx mice leads to new muscle tissue and dystrophin expression, whole BM cells and BM derived myogenic cells from male wild-type mice were injected into the tail vein of sublethally irradiated female mdx mice. Twelve weeks after transplantation the Tibialis anterior muscles were analyzed for dystrophin expression by immunocytochemistry and FISH analysis using a Y-chromosome-specific sequence to detect donor derived male cell. The proportion of donor derived dystrophin-positive fibers was 0,5-2%
  
• Neuroectodermal differentiation of BM stem cells
  In previous studies using intravenous whole BM transplantation in mice, we evaluated the incorporation of BM cells in murine brain, spinal cord and ganglia. (Corti et al. 2002). We investigated whether the expansion and mobilization of circulating BM stem cells by in-vivo treatment with Granulocyte-Colony Stimulating Factor (G-CSF) and Stem Cell Factor (SCF) increased the amount of BM-derived neuronal cells in mouse brain. We transplanted adult BM from transgenic GFP mice into lethally irradiated adults and newborns. Three months after transplantation the mean degree of BM chimerism was 70%. GFP+- donor-derived Y chromosome positive cells (as observed by FISH) and Y- were present in hematopoietic compartments and were detected in several brain areas of all treated mice (cortical and subcortical areas, cerebellum, OB). To evaluate whether GFP+ cells have acquired a neuroectodermal phenotype we analyzed the coexpression of GFP and neuronal markers by confocal analysis. A proportion of these cells, within the neural cortex, spinal cord and sensory ganglia co-expressed several neuronal markers like NeuN, NF, TuJ1, MAP-2. These data confirm that BM-derived cells may migrate and reside into the CNS. Twenty percent of GFP+ cells had a ramified shape, and were positive for Mac-1 and F4/80, two markers expressed exclusively on macrophages and microglia. We concluded that these cells are microglia and that BM derived cells contribute to microglial genesis. The presence of GFP+ cells expressing NeuN, NF and TuJ1 in cortical forebrain and OB was higher in G-CSF-SCF treated groups (p<0.05, analysis of variance, Fisher post hoc). We observed that overall the amount of double positive cells was higher in animals treated at birth than in adults, and in OB than in forebrain areas (p<0.05). Our results indicated that G-CSF and SCF administration modulates the availability of GFP+ cells in the brain and enhances their capacity to acquire neuronal characteristics. Cytokine stimulation of autologous stem cells might be seen as a new strategy for neuronal repair in neurodegenerative diseases