Plant a Tree
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See our Returns Policy. Visit our Help Pages. Audible Download Audio Books. Shopbop Designer Fashion Brands. Amazon Prime Music Stream millions of songs, ad-free. S1 B , although a certain level of variability was noticed. We also performed western blot analysis on the same samples with antibodies specific to superoxide dismutase 1 and Catalase, but again no significant differences were identified not shown.
We derived neural precursors from hair bulge whisker Supplementary Material, Fig. S2 A and sciatic nerve Supplementary Material, Fig. Cells showed typical morphology of neural crest precursor Supplementary Material, Fig. S2 C and D and were negative for glial fibrillary acidic protein staining Supplementary Material, Fig. S2 E and F. After induction with suitable culture medium, differentiated neurons Supplementary Material, Fig.
S2 I—L were obtained. Mitochondrial morphology evaluated with the Mitotracker-red. F Control fibroblasts treated with valinomycin. This parameter resulted in a 2. We observed that the Mitotracker stains mitochondria in untreated cells Fig. The JC1 dye undergoes a reversible change in fluorescence emission from green to red as mitochondrial membrane potential increases. Cells with high membrane potential promote the formation of red fluorescent aggregates, while cells with low membrane potential show a diffuse green fluorescence.
JC1 specificity was tested by pre-treating WT neurons with valinomycin. As reported in Supplementary Material, Figure S3 , treated neurons stain green fluorescent aggregates, whereas untreated neurons showed red fluorescent aggregates. A and B JC1 staining of neurons derived from hair bulge. C and D JC1 staining of neurons derived from sciatic nerve. E and F JC1 staining of neonatal neuron derived from hippocampus. Blue fluorescence indicates DAPI staining of nuclei. G and H Electron microscopy of neurons derived from sciatic nerve semithin section Toluidin Blue.
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I — L Electron microscopy of neurons derived from sciatic nerve ultrastructure uranyl acetate and lead citrate. To corroborate our observations, we performed electron microscopic analysis on neurons in culture, which were stained with JC1. To this aim, we derived neonatal hippocampal neonatal neurons and we performed the JC1 staining experiment.
Altogether, these data demonstrated that aberrant mitochondria were present not only in neurons derived from the sciatic nerve of adult animals, but also in neonatal hippocampal neurons. Examination of semithin sections, counterstained with Toluidine Blue, revealed no obvious differences in myelinated fibre density, in the absence of demyelinating features.
Overall, no clear-cut sign of progressive neuropathy was present, since no reduction in myelinated fibres or increase in the number of degenerating fibres has been detected Fig. Evaluation of neurodegeneration of peripheral nervous systems with optic and electron microscopy. However, a substantial number of myelinated fibres Fig. S4 B—D swollen mitochondria, characterized by alteration of cristae and by the presence of multivescicolar bodies or amorphous material, in the matrix Fig.
Electron microscopy of peripheral and CNS of and week-old animals. We also analysed the basal ganglia of week-old mice. These data indicate the presence of defective mitochondria in both the peripheral and CNSs in mice with defective Pank2. We have demonstrated that the murine Pank2 protein is mainly located in the mitochondrial inter-membrane space, which is compatible with its role in CoA metabolism and with its regulation in the presence of CoA at concentrations found in mitochondrial matrix Further investigation using, for instance, immuno-electronmicroscopy is needed to unequivocally establish this sub-mitochondrial localization.
In fact, although the mouse model does not recapitulate the clinical and neuropathological features of the human condition 7 , it could serve as a system in which to interrogate the basic defect in mitochondrial function. However, recent observations indicate that mitochondrial respiration is largely carried out by the fraction of respiratory chain complexes that assemble together in supercomplexes forming functionally active units, called respirasomes 15 , Thus, the spectrophotometric measurement of each respiratory complex activity does not necessarily reflect the mitochondrial functional capacity in vivo.
Together, these results indicate that the absence of the Pank2 protein leads to a global failure of the mitochondrial bioenergetic performance without affecting the function of any single respiratory chain complex. An interesting result of our study, strictly correlated with the absence of Pank2 function, was the demonstration of alteration in mitochondrial membrane potential in neurons derived from sciatic nerve and hair bulge stem cells of adult mice.
Recently, the characterization of a PKAN Drosophila model demonstrated that impaired function of pantothenate kinase induced a neurodegenerative phenotype with mitochondrial dysfunction, decreased levels of CoA, increased protein oxidation and reduced lifespan It is tempting to speculate that the reduction in the respiration could be attributable to the severe alteration of the cristae structure, which could prevent the respirasomes from remaining functionally active.
As demonstrated by other studies, mitochondrial membrane damage contributes to the pathogenesis of many neurodegenerative diseases 19 , Interestingly enough, a recent investigation of a KO mouse model for the Pla2g6 gene, which is defective in a different but related form of NBIA, revealed the presence of collapsed mitochondria with degenerated inner membranes Pla2g6 gene encodes a group VIA calcium-independent phospholipase A2, an esterase that hydrolyzes the sn-2 ester bond in phospholipids to yield free fatty acids and lysophospholipids and which is involved in cardiolipin remodelling Analysis of phospholipids and fatty acids revealed differences between KO and WT mice.
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The vulnerability of mitochondrial inner membranes in Pla2g6 KO mice might be attributable to increased production of reactive oxygen species ROS 23 and a rich content of polyunsaturated fatty acids that can readily be peroxidized, such as linoleic acid in cardiolipin Alteration in cholesterol and lipid metabolism was also recently demonstrated by a metabolomics investigation in a group of PKAN patients In this case, however, the alteration was mainly due to a defective synthesis and not, as in the case of PLA2G6, to the absence of a catabolic enzyme.
Irrespective of the anabolic PANK2 or catabolic PLA2G6 role of these two mitochondrial proteins, a common culprit in the pathogenesis of both neurodegenerative diseases could be an altered lipid metabolism It is possible that mice can better tolerate alterations in bioenergetics metabolism without suffering any overt clinical manifestations because of the presence of compensatory mechanisms.
Few examples of mouse models of mitochondrial disorders, which display a biochemical phenotype but do not present any clinical signs typical of the human pathology, are described in the literature 26 , However, our findings suggest that there would be value in investigating bioenergetic competence and ultra-structural abnormalities of mitochondria in PKAN patients.
The use and care of animals followed the Italian Law D.
Animal studies were approved in accordance with guidelines of the Italian Ministry of Health: Standard diet DS, Mucedola, Italy and water were given ad libitum. Standard methods were used for the preparation of mitochondrial and post-mitochondrial fractions from mouse brain Brain was extracted and rinsed using cold homogenization medium AT [0. The organ weighted was cut into small pieces and washed to remove blood and connective tissue.
The brain was homogenized with 10—15 strokes using a Dounce-type glass homogenizer with a manually driven glass pestle, adding 5 ml of homogenization medium AT per gram of the starting material. The supernatant obtained from this centrifugation contained cytosol. To clean the mitochondrial pellet obtained, we resuspended it with 5 ml of medium AT, transferred to 1. This fraction contained free mitochondria and synaptosomes. Mitoplasts were then sonicated 10 s for three times at 10 Amp.
Thirty micrograms of proteins were used for each sample in denaturing sodium dodecyl sulphate—polyacrylamide gel electrophoresis SDS—PAGE.
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Western blot analysis was performed as described 29 , using the ECL-chemiluminescence kit Amersham. The following mouse monoclonal antibodies were used: An anti-ethylmalonic encephalopathy 1 rabbit polyclonal antibody was used at 1: Secondary anti-rabbit and anti-mouse antibodies were used at 1: Oxidized proteins were detected using the Oxyblot Protein Oxidation Detection Kit Millipore following the manufacturer's instructions.
Brain extracts were homogenized in 20 m m Tris—HCl, pH 7, 50 m m dithiothreitol in the presence of anti-protease inhibitor cocktail tablet Roche and then derivatized to 2,4-dinitrophenylhydrazone. These data were calculated in 66 neurons 33 KO versus 33 WT in three different experiments. Detection of mitochondrial potential change was performed using JC1 Sigma, CS staining kit according to manufacturer' instructions. Measurements of oxygen consumption were performed on isolated mitochondria derived from mouse brain using an XF96 Extracellular Flux Analyser Seahorse Bioscience.
To rapidly isolate mitochondria, brains were obtained from sacrificed mice and washed three times in ice-cold phosphate-buffered saline PBS , than homogenized with 15 strokes in a glass-dounce homogenizer with 2 ml of hepes, EDTA, sucrose HES —0. The supernatant was collected and centrifuged for 15 min at g and the supernatant was discarded; the pellet was resuspended in HES—0. The pellet containing mitochondria was resuspended in HES—0. As indicated by Rogers et al. OCR was also measured after addition of carbonyl cyanide 4- trifluoromethoxy phenylhydrazone FCCP , to evaluate the maximal respiratory capacity.
Although the measurements obtained from the replicates for each sample were relatively consistent in individual experiments, the values may vary in different experiments performed in different days. In practice, for each sample, the overall mean was subtracted from each measure, and the result was divided by the overall SD of the experiment.
The resulting values indicate how many SD's separate a given observation from the reference value of the experiment. Transformed data were then compared across samples and across experiments by unpaired two-tailed Student's t -test. One-millimetre-thick sections were stained with Toluidine Blue. For ultra-structural analysis, sections of 90 nm were collected on mesh copper grids, positively stained with uranyl acetate and lead citrate, and then analysed by transmission electron microscope EM Zeiss to evaluate the presence of possible alterations in axonal morphology, cell membranes and mitochondria.
Selected areas of interest were sampled, post-fixed in osmium tetroxide and embedded in Epon epoxy resin. Thin sections 80—90 nm were stained with uracyl acetate and lead citrate and examined with a CM10 Philips electron microscope.