Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Summary

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Abstract

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan. Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays.

Protocol

Nuclei Extraction

1. Put 250 mg of frozen postmortem brain tissue in 5 mL Lysis Buffer¹ in a douncer and place it on ice. To receive about 5 million nuclei after FACS sorting, we usually use 1000 mg of tissue per sample.
2. Once the tissue has thawed, dounce the tissue for 1 minute while on ice.
3. Take the homogenized tissue and place it into a 15 mL clear ultracentrifuge tube. Put the tube on ice.
4. Take 9 mL of Sucrose Solution² with a pipette, put the pipette at the bottom of the clear ultracentrifuge tube, and release the Sucrose Solution² to create a concentration gradient with the homogenized tissu....

Discussion

The protocol presented here should be particularly useful for studies focused on epigenetic changes of neuronal chromatin and, more generally, on the molecular phenotype of the neuronal nucleus, during normal developing and aging, or in neurological or psychiatric disease. The method is of particular advantage when the cellular heterogeneity of brain tissue, including potential shifts in neuron-to-glia ration during the course of aging or due to disease are a concern ¹. For example, by combining the present so.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents: 1. Lysis Buffer 0.32M Sucrose 5.47 g 5 mM CaCl2 250 μl 3 mM Mg(Acetate)2 150 μl 0.1 mM EDTA 10 μl 10mM Tris-HCl, pH8 500 μl 1 mM DTT 17 μl 0.1% Triton X-100 50 μl Adjust volume to 50 ml with Autoclaved water 2. Sucrose Solution 1.8 M Sucrose 30.78 g 3 mM Mg(Acetate)2 150 μl 1 mM DTT 17 μl 10 mM Tris-HCl, pH8 500 μl Adjust volume to 50 ml with Autoclaved water 3. Dounce buffer 10 mM Tris 0.242 g 4 mM MgCl2 0.163 g 1 mM CaCl2 0.03 g Adjust pH to 7.5 and volume to 200 ml with Autoclaved water