Aging-dependent functional alterations of mitochondrial DNA (mtDNA) from human fibroblasts transferred into mtDNA-less cells

To investigate the role that aging-dependent accumulation of mitochondrial DNA (mtDNA) mutations plays in the senescence processes, mitochondria from fibroblasts of 21 normal human individuals between 20 weeks (fetal) and 103 years of age were introduced into human mtDNA-less (ρ0) 206 cells by cytoplast × ρ0 cell fusion, and 7-31 transformant clones were isolated from each fusion. A slight cell donor age-dependent decrease in growth rate was detected in the transformants. Using an O2 consumption rate of 1 fmol/min/cell, which was not observed in any transformant among 158 derived from individuals 20 weeks (fetal) to 37 years of age, as a cut-off to identify respiratory-deficient clones, 11 such clones were found among 198 transformants derived from individuals 39-103 years of age. Furthermore, conventional and nonparametric analysis of the respiratory rates of 356 clones revealed a very significant decrease with donor age. In other analyses, a very significant age-dependent decline in the mtDNA content of the clones was observed, without, however, any significant correlation with the decrease in O2 consumption rate in the defective transformants. These observations clearly indicate the occurrence in the fibroblast-derived transformants of two independent, age-related functional alterations of mtDNA, presumably resulting from structural damage to this genome

Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-alpha and interleukin-8 mRNA in human neutrophils. Evidence for a role of L-selectin as a signaling molecule.

Sulfatides have been established recently as ligands for L-selectin, and we investigated whether they trigger transmembrane signals through ligation of L-selectin. We found that sulfatides trigger the increase of cytosolic free calcium in neutrophils and that this effect was strictly dependent on sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory. Chymotrypsin and phorbol 12-myristate 13-acetate treatment of neutrophils caused shedding of L-selectin, but not of class I major histocompatibility complex antigens or beta 2 integrins, and blunted the capability of neutrophils to respond to sulfatides with an increase of cytosolic free calcium. Four different anti-L-selectin antibodies (DREG-200, LAM1/3, LAM1/6, and LAM1/10), but not four control antibodies directed against different surface molecules of neutrophils, also triggered an increase of cytosolic free calcium. The anti-L-selectin antibodies were stimulatory both if used in a soluble form, after cross-linking with anti-mouse F(ab')2 fragments, and immobilized to protein A of Staphylococcus aureus through the Fc fragment. With immobilized antibodies, an increase of cytosolic free calcium was found also by plating neutrophils on antibodies bound to protein A-coated coverslips and monitoring the increase of cytosolic free calcium by fluorescence microscopy. Both sulfatides and anti-L-selectin antibody effects were not inhibited by pertussis toxin, thus indicating that a pertussis toxin-sensitive GTP-binding protein was not involved in signal transduction. Sulfatides also triggered an increase of tumor necrosis factor-alpha and interleukin-8 mRNAs in neutrophils. Also to act as stimuli of cytokine mRNA expression, sulfatides required sulfation of the galactose ring, as non-sulfated galactocerebrosides were not stimulatory, and depended on expression of L-selectin, as shedding of this molecules induced by chymotrypsin blunted their effects. These findings suggest that L-selectin can transduce signals activating selective cell function

Alternative sources of neurons and glia from somatic stem cells.

Stem cell populations have been shown to be extremely versatile: they can generate differentiated cells specific to the tissue in which they reside and descendents that are of different germ layer origin. This raises the possibility of obtaining neuronal cells from new biological source of the same adult human subjects. In this study, we found that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cell-like population isolated from several newborn and adult mouse tissues: muscle and hematopoietic tissues. This population, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Brain engraftment of the somatic-derived neural stem cells generated neuronal phenotypes, demonstrating the great plasticity of these cells with potential clinical application

In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation.

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to β-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of β-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cellmediated gene therapy of inherited muscle disorders

Intraarterial Injection of Muscle-Derived Cd34+Sca-1+ Stem Cells Restores Dystrophin in mdx Mice

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% β-galactosidase–positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage

Aging-Dependent Large Accumulation of Point Mutations in the Human mtDNA Control Region for Replication

Progressive damage to mitochondrial DNA (mtDNA) during life is thought to contribute to aging processes. However, this idea has been difficult to reconcile with the small fraction of mtDNA so far found to be altered. Here, examination of mtDNA revealed high copy point mutations at specific positions in the control region for replication of human fibroblast mtDNA from normal old, but not young, individuals. Furthermore, in longitudinal studies, one or more mutations appeared in an individual only at an advanced age. Some mutations appeared in more than one individual. Most strikingly, a T414G transversion was found, in a generally high proportion (up to 50 percent) of mtDNA molecules, in 8 of 14 individuals above 65 years of age (57 percent) but was absent in 13 younger individuals

Central nervous system trans-synaptic effects of acute axonal injury : a 1-H magnetic resonance spectroscopy study

N-acetylaspartate (NAA) has previously been proposed as a neuronal marker. 1H magnetic resonance spectroscopy (MRS) is able to detect NAA in brain, and decreases of NAA have been documented after brain injury. The reason for this decrease is not fully understood and neuron loss damage and "dysfunction" have all been proposed. It is hypothesized that acute central nervous system (CNS) deafferentation causes a trans-synaptic NAA decrease and that high resolution 1H MRS is able to detect such a decrease. To test this hypothesis, an experimental model was used in which axonal lesions were obtained by stretch injury in guinea pig right optic nerve (95-99% crossed fibers). The trans-synaptic concentration of NAA, total creatine (Cr), and the NAA/Cr ratio in lateral geniculate bodies (LGB) and superior colliculi (SC) sample extracts were measured 72 h later by high resolution 1H MRS. In the left LGB/SC, which is where right optic nerve fibers project, reductions of NAA and NAA/Cr were found whereas Cr levels were normal. NAA, NAA/Cr, and Cr values were all normal in the right LGB/SC. Histology and EM findings revealed no abnormalities. At 7 days, left LGB/SC NAA and NAA/Cr values were in the normal range. It was concluded that 1) acute deafferentation in the CNS causes a trans-synaptic decrease of NAA levels that can be detected by 1H MRS and 2) NAA decrease may be due to changes of NAA metabolism caused by functional neuronal inactivity rather than neuronal loss, injury or "dysfunction." 1H MRS is a potential tool for the study of functional effect of CNS lesions in vivo