Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment

Tenreiro, P.; Rebelo, Sandra; Martins, Filipa; Santos, Mariana; Coelho, Elisabete; Almeida, Miguel; Matos, A. P. Alves de; Silva, Edgar F. da Cruz e

doi:10.1016/j.ab.2016.10.015

Cited by 18 publications

(10 citation statements)

References 29 publications

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“…Myelin and synaptosome isolation Myelin and synaptosomes were isolated from adult mouse brains using a sucrose gradient as previously described (Tenreiro et al, 2017). Mouse brains were homogenized with a Dounce glass tissue grinder in cold homogenization buffer (5mM HEPES(pH 7.4), 1mM MgCl2, 0.5mM CaCl2) and resuspended in 0.32M Tris-buffered sucrose.…”

Section: Methods Detailsmentioning

confidence: 99%

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination

Shen¹,

Reichelt²,

Kyauk³

et al. 2021

Cell Reports

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Section: Methods Detailsmentioning

confidence: 99%

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination

Shen¹,

Reichelt²,

Kyauk³

et al. 2021

Cell Reports

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“…The advantages of Percoll include low viscosity, shorter centrifugation time, and maintaining constant osmolarity to prevent shrinkage/expansion [41]. However, synaptosomes collected from Percoll gradients (at 10/20% interface) are less enriched in synaptic terminals than those collected with sucrose gradients [42]. There is a tradeoff between synaptosome integrity and purity.…”

Section: Isolation Of Human Synaptosomesmentioning

confidence: 99%

The Study of Postmortem Human Synaptosomes for Understanding Alzheimer’s Disease and Other Neurological Disorders: A Review

Jhou

Tai

2017

Neurol Ther

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Synaptic dysfunction is thought to play important roles in the pathophysiology of many neurological diseases, including Alzheimer’s disease, Parkinson’s disease, and schizophrenia. Over the past few decades, there have been systematic efforts to collect postmortem brain tissues via autopsies, leading to the establishment of dozens of human brain banks around the world. From cryopreserved human brain tissues, it is possible to isolate detached-and-resealed synaptic terminals termed synaptosomes, which remain metabolically and enzymatically active. Synaptosomes have become important model systems for studying human synaptic functions, being much more accessible than ex vivo brain slices or primary neuronal cultures. Here we review recent advances in the establishment of human brain banks, the isolation of synaptosomes, their biological activities, and various analytical techniques for investigating their biochemical and ultrastructural properties. There are unique insights to be gained by directly examining human synaptosomes, which cannot be substituted by animal models. We will also discuss how human synaptosome research has contributed to better understanding of neurological disorders, especially Alzheimer’s disease.

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“…They remain able to store, release, and take up neurotransmitters. In addition, synaptosomes can be isolated from post mortem human nervous tissue subserving the molecular analysis of synaptopathies underlying human neurological and psychiatric disorders, and are starting points for further subcellular fractionation processes for the isolation of synaptic vesicles, presynaptic detergent soluble membrane fraction or the postsynaptic density (Dunkley et al 2008;Tenreiro et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Proteomic comparison of different synaptosome preparation procedures

et al. 2020

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Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

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Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment

Cited by 18 publications

References 29 publications

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination

The Study of Postmortem Human Synaptosomes for Understanding Alzheimer’s Disease and Other Neurological Disorders: A Review

Proteomic comparison of different synaptosome preparation procedures

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