Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons

Summary

We have developed a reliable method of selective plasma membrane permeabilization of primary mouse cortical neurons for high content automated analysis of neuronal nucleocytoplasmic transport.

Abstract

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

Introduction

Disruption of nucleocytoplasmic transport, the regulated trafficking of proteins and RNA between the nucleus and cytoplasm, is increasingly implicated in the pathogenesis of neurodegenerative diseases (recently reviewed¹). We and others have reported structural and functional disruption of the nucleocytoplasmic transport apparatus in postmortem tissue and animal models of C9orf72 amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), Alzheimer’s disease, and Huntington's disease^2,3,4,5

Protocol

First, the protocol describes the generation of primary neuronal cultures (step 1) and preparation of materials for the transport assay (step 2), followed by the transport assay itself (steps 3-4) and image acquisition and analysis (step 5). All methods described here were approved by the Animal Care and Use Committee (ACUC) of Johns Hopkins University.

1. Primary mouse cortical neuron cultures

1. Prepare the stock solutions.
   1. Dissection buffer: Combine 50 mL of 10x HBSS, 1.......

Discussion

The protocol detailed above provides a reliable and reproducible method for selectively permeabilizing the plasma membrane of primary mouse cortical neurons for nuclear import analysis. Here, an application of the method for nuclear import analysis of a direct importin β cargo (Rango) is shown, but this same approach can be used to analyze the passive and active import of a wide range of fluorescent cargoes. Permeabilization enables precise manipulation of the transport reaction in ways that are not feasible in inta.......

Materials

Name	Company	Catalog Number	Comments
1 M HEPES	Gibco	15630-080
10x HBSS	Gibco	14185-052
32% paraformaldehyde	Electron Microscopy Sciences	15714-S
96-well optical glass plates	CellVis	P96-1.5H-N
ATP lithium salt	Millipore Sigma	11140965001
B27	Gibco	17504-044
Bio-Rad Protein Assay Kit II	Bio-Rad	5000002
BL21(DE3) E. coli	NEB	C2527H
Bovine serum albumin fraction V, heat shock, fatty acid free	Sigma-Aldrich	3117057001
Chromatography columns	Bio-Rad	7311550
Creatine kinase	Millipore Sigma	10127566001
Creatine phosphate	Millipore Sigma	10621722001
Dextran, Texas Red, 70,000 MW	Thermo Fisher	D1864
DNase I	Sigma-Aldrich	DN25
E15-16 timed pregnant C57BL/6J female mice	Jackson Laboratory	000664
Excel	Microsoft	N/A
Fetal bovine serum	Hyclone	SH30070.03
Glutamax	Gibco	35050-061
Glycerol	Thermo Fisher	15514011
GTP lithium salt	Millipore Sigma	11140957001
HALT protease inhibitor (100x)	Thermo Fisher	78439
HEK293T cells	ATCC	CRL-3216
HIS-Select HF Nickel affinity gel	Sigma-Aldrich	HO537
Hoechst 33342	Thermo Fisher	H1399
ImageExpress Micro Confocal High-content Imaging System	Molecular Devices	N/A	Used for time-lapse imaging
Imidazole	Millipore	I3386
Importazole	Sigma-Aldrich	SML0341
IPTG	Corning	46-102-RF
Laminin	Sigma-Aldrich	L2020
LB broth	Grainger	31FZ62
LSM800 confocal microscope	Zeiss	N/A	Used for dextran imaging
MetaXpress High Content Image Analysis Software	Molecular Devices	N/A
Neurobasal medium	Gibco	21103
Papain	Worthington	LS003126
Penicillin-streptomycin	Gibco	15140-122
Poly-D-Lysine	Sigma-Aldrich	P6407
Protease inhibitor cocktail	Millipore Sigma	11873580001
Rango Plasmid (pRSET Rango2/a1 + linkers)	N/A	N/A	pK44, containing N-terminal 6-His tag
SOC (super optimal broth with catabolite repression) media	Quality Biological	340-031-671
Sodium pyruvate	Gibco	11360-070
Spin-X UF concentrators (30K MWCO)	Corning	CLS431484
Trypsin-EDTA (0.05%)	Gibco	25300054