Isolation of Myelin

Larocca, Jorge N.; Norton, Williams T.

doi:10.1002/0471143030.cb0325s33

Cited by 85 publications

(79 citation statements)

References 26 publications

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“…Because we have excluded contamination with lysosomes and peroxisomes, it is more likely that those vesicles are remnants of cytoplasmic membranes. Myelin consists of 80% lipids and 20% proteins; therefore, a purification step with Percoll gradient eliminates the possibility of contamination of the final mitochondrial fraction with myelin (21). Figure 5 also shows that there were no apparent contaminants resembling myelin in the pellets of BM and SCM.…”

Section: Respiratory Activities Of the Spinal Cord And Brain Mitochonmentioning

confidence: 90%

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

Panov

Kubalik

Zinchenko

et al. 2011

American Journal of Physiology-Regulatory, Integrative and Comp

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Mitochondrial dysfunctions contribute to neurodegeneration, the locations of which vary among neurodegenerative diseases. To begin to understand what mechanisms may underlie higher vulnerability of the spinal cord motor neurons in amyotrophic lateral sclerosis, compared with brain mitochondria, we studied three major functions of rat brain mitochondria (BM) and spinal cord mitochondria (SCM) mitochondria: oxidative phosphorylation, Ca 2ϩ sequestration, and production of reactive oxygen species (ROS), using a new metabolic paradigm (Panov et al., J. Biol. Chem. 284: 14448 -14456, 2009). We present data that SCM share some unique metabolic properties of the BM. However, SCM also have several distinctions from the BM: 1) With the exception of succinate, SCM show significantly lower rates of respiration with all substrates studied; 2) immunoblotting analysis showed that this may be due to 30 -40% lower contents of respiratory enzymes and porin; 3) compared with BM, SCM sequestered 40 -50% less Ca 2ϩ , and the total tissue calcium content was 8 times higher in the spinal cord; 4) normalization for mitochondria from 1 g of tissue showed that BM can sequester several times more Ca 2ϩ than was available in the brain tissue, whereas SCM had the capacity to sequester only 10 -20% of the total tissue Ca 2ϩ ; and 5) with succinate and succinate-containing substrate mixtures, SCM showed significantly higher state 4 respiration than BM and generated more ROS associated with the reverse electron transport. We conclude that SCM have an intrinsically higher risk of oxidative damage and overload with calcium than BM, and thus spinal cord may be more vulnerable under some pathologic conditions. (250) oxidative phosphorylation; permeability transition; reactive oxygen species MITOCHONDRIAL DYSFUNCTIONS play important roles in pathogenesis of degenerative diseases of the central nervous system (4,14). Although many neurodegenerative diseases are systemic, such as Huntington's disease (15), amyotrophic lateral sclerosis (ALS) (2), and Friedrich's ataxia (8), neurodegeneration predominantly occurs at specific locations within the central nervous system in each of these diseases. An important question in neurodegenerative diseases is what determines localization of neuronal death? There is evidence that, during systemic intoxication with rotenone, or in transgenic mice bearing mutated human G93A SOD1 gene, the pathological changes occur in brain and spinal cord mitochondria, but not in liver mitochondria (10, 32). It was also shown that various brain regions differ in their sensitivity to the deleterious effects of oxygen deprivation (22, 41). Thus, differences in the tissue-specific and regional properties of mitochondria may contribute to selectivity of neurodegeneration.Regarding ALS, one of the major questions is, "Why are motor neurons in the spinal cord more vulnerable than neurons in brain?" Because mitochondrial dysfunctions play important roles in pathogenesis of ALS (2, 24), the above question can be restated as, "What featu...

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Section: Respiratory Activities Of the Spinal Cord And Brain Mitochonmentioning

confidence: 90%

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

Panov

Kubalik

Zinchenko

et al. 2011

American Journal of Physiology-Regulatory, Integrative and Comp

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“…A light-weight membrane fraction enriched for myelin was purified from mouse sciatic nerves homogenized in 0.27 M sucrose according to Larocca and Norton (2007). The protein concentration was determined using the 2-D Quant kit according to the manufacturer (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Quantitative and Integrative Proteome Analysis of Peripheral Nerve Myelin Identifies Novel Myelin Proteins and Candidate Neuropathy Loci

Patzig¹,

Jahn²,

Tenzer³

et al. 2011

J. Neurosci.

157

190

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Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia-axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relative abundance was previously misestimated due to technical limitations regarding protein separation and visualization. Focusing on tetraspan-transmembrane proteins, we validated novel myelin constituents using immunobased methods. Bioinformatic comparison with mRNA-abundance profiles allowed the categorization in functional groups coregulated during myelin biogenesis and maturation. By differential myelin proteome analysis, we found that the abundance of septin 9, the protein affected in hereditary neuralgic amyotrophy, is strongly increased in a novel mouse model of demyelinating neuropathy caused by the loss of prion protein. Finally, the systematic comparison of our compendium with the positions of human disease loci allowed us to identify several candidate genes for hereditary demyelinating neuropathies. These results illustrate how the integration of unbiased proteome, transcriptome, and genome data can contribute to a molecular dissection of the biogenesis, cell biology, metabolism, and pathology of myelin. Peripheral nerve myelin facilitates rapid impulse conduction and normal motor and sensory functions. Many aspects of myelin biogenesis, glia-axonal interactions, and nerve homeostasis are poorly understood at the molecular level. We therefore hypothesized that only a fraction of all relevant myelin proteins has been identified so far. Combining gel-based and gel-free proteomic approaches, we identified 545 proteins in purified mouse sciatic nerve myelin, including 36 previously known myelin constituents. By mass spectrometric quantification, the predominant P0, periaxin, and myelin basic protein constitute 21, 16, and 8% of the total myelin protein, respectively, suggesting that their relative abundance was previously misestimated due to technical limitations regarding protein separation and visualization. Focusing on tetraspan-transmembrane proteins, we validated novel myelin constituents using immuno-based methods. Bioinformatic comparison with mRNA-abundance profiles allowed the categorization in functional groups coregulated during myelin biogenesis and maturation. By differential myelin proteome analysis, we found that the abundance of septin 9, the protein affected in hereditary neuralgic amyotrophy, is strongly increased in a novel mouse model of demyelinating neuropathy caused by the loss of prion pr...

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“…In addition, UV-absorbing components within the lipid rich myelin obscure the polyribosome profiles. Myelin flotation is therefore necessary, where dense compounds such as polyribosomes are pelleted and lighter compounds such as myelin float and are removed 16 . Polyribosomes are then sedimented over a 17.5% to 50% sucrose gradient.…”

Section: Discussionmentioning

confidence: 99%

<em>In vivo</em> Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Lou

Baser

Klußmann

et al. 2014

JoVE

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Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting 'translatomics' to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

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Isolation of Myelin

Cited by 85 publications

References 26 publications

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

Quantitative and Integrative Proteome Analysis of Peripheral Nerve Myelin Identifies Novel Myelin Proteins and Candidate Neuropathy Loci

<em>In vivo</em> Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

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