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Isolation of lysosomes from brain tissue. A separation method by means of alteration of mitochondrial and synaptosomal sedimentation properties.

ABSTRACT: 1. A crude mitochondrial-lysosomal preparation from brain tissue was layered on a sucrose gradient containing 20mm-succinate, 10mm-Tris and 1mm-disodium EDTA at pH7.4. The lysosomes were separated from the mitochondria and synaptosomes by means of a twosteps centrifugation procedure. In a first low-speed step (40min at 5300g at 15 degrees C) the sedimentation rate of mitochondria and mitochondria-containing synaptosomes was enlarged due to passage of these subcellular structures through the sucrose gradient with the above-mentioned chemicals (called ;chemical field'). That part of the gradient which contained the mitochondria and synaptosomes was removed and substituted by a gradient suitable for isopycnic isolation of lysosomes in a second centrifugation step. The achieved purification for bovine brain lysosomes was 5-8-fold, for rat brain lysosomes 7-10-fold, over the homogenate. 2. The enlargement of the sedimentation rate of mitochondria and synaptosomes was brought about by the presence of succinate, but also by one of the following salts in the chemical field: malonate, fumarate, pyruvate, phosphate and chloride. 3. Comparison of the chemical-field method with other methods for the isolation of lysosomes showed that (a) with the chemical-field method a 2-3-times higher purification of the rat and bovine brain lysosomal fraction can be achieved than with the procedure described by Koenig, Gaines, McDonald, Gray & Scott [(1964) J. Neurochem.11, 729-743], and that (b) similar purification results for rat liver lysosomes were obtained when the chemical-field method and the procedure described by van Dijk, Roholl, Reijngoud & Tager [(1976) FEBS Lett.62, 177-181] were compared.

SUBMITTER: Lisman JJ

PROVIDER: S-EPMC1186482 | biostudies-other | 1979 Jan

REPOSITORIES: biostudies-other

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Project description:Oxidative stress is frequently implicated in the pathology of neurodegenerative disease. The chief source of this stress is mitochondrial respiration, via the passage of reducing equivalents through the respiratory chain resulting in a small but potentially pathological production of superoxide. The superoxide that is produced during normal respiration is primarily detoxified within the mitochondria by superoxide dismutase 2 (Sod2), a key protein for maintaining mitochondrial function. Mitochondria are distributed throughout the soma of neurons, as well as along neuronal processes and at the synaptic terminus. This distribution of potentially independent mitochondria throughout the neuron, at distinct subcellular locations, allows for the possibility of regional subcellular deficits in mitochondrial function. There has been increasing interest in the quantification and characterization of messages and proteins at the synapse, because of its importance in neurodegenerative disease, most notably Alzheimer disease. Here, we report the transcriptomic and proteomic changes that occur in synaptosomes from frontal cortices of Sod2 null mice. Constitutively Sod2 null mice were differentially dosed with the synthetic catalytic antioxidant EUK-189, which can extend the life span of these mice, as well as uncovering or preventing neurodegeneration due to endogenous oxidative stress. This approach facilitated insight into the quantification of trafficked messages and proteins to the synaptosome. We used two complementary methods to investigate the nature of the synaptosome under oxidative stress: either whole-genome gene expression microarrays or mass spectrometry-based proteomics using isobaric tagging for relative and absolute quantitation of proteins. We characterized the relative enrichment of gene ontologies at both gene and protein expression levels that occurs from mitochondrial oxidative stress in the synaptosome, which may lead to new avenues of investigation in understanding the regulation of synaptic function in normal and diseased states. As a result of using these approaches, we report for the first time an activation of the mTOR pathway in synaptosomes isolated from Sod2 null mice, confirmed by an upregulation of the phosphorylation of 4E-BP1.

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Isolation of lysosomes from brain tissue. A separation method by means of alteration of mitochondrial and synaptosomal sedimentation properties.

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