Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration

Summary

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

Abstract

The nuclear pore complex (NPC) is a complex macromolecular structure comprised of multiple copies of ~30 different nucleoporin proteins (Nups). Collectively, these Nups function to regulate genome organization, gene expression, and nucleocytoplasmic transport (NCT). Recently, defects in NCT and alterations to specific Nups have been identified as early and prominent pathologies in multiple neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD)/Frontotemporal Dementia (FTD), and Huntington's Disease (HD). Advances in both light and electron microscopy allow for a thorough examination of sub-cellular structures, including the NPC and its Nup constituents, with increased precision and resolution. Of the commonly used techniques, super-resolution structured illumination microscopy (SIM) affords the unparalleled opportunity to study the localization and expression of individual Nups using conventional antibody-based labeling strategies. Isolation of nuclei prior to SIM enables the visualization of individual Nup proteins within the NPC and nucleoplasm in fully and accurately reconstructed 3D space. This protocol describes a procedure for nuclei isolation and SIM to evaluate Nup expression and distribution in human iPSC-derived CNS cells and postmortem tissues.

Introduction

The prevalence of age-related neurodegenerative diseases is increasing as the population ages¹. While the genetic underpinnings and pathologic hallmarks are well characterized, the precise molecular events leading to neuronal injury remain poorly understood^{2,3,4,5,6,7,8,9,10,11,12}

Protocol

All blood samples for iPSC generation and autopsied tissue collections are approved by Johns Hopkins IRB with Johns Hopkins ethics oversight. All patient information is HIPPA compliant. The following protocol adheres to all Johns Hopkins biosafety procedures.

1. Preparation of slides for immunostaining and imaging

1. Position a positively charged glass microscope slide in an empty cytofunnel and draw a circle with a hydrophobic barrier pen to outline an area to deposit the nuclei.
2. Add 50 μL of 1 mg/mL collagen solution (diluted in 1x PBS) to the center of the circle drawn in step 1.1 and incubate at room temperature ....

Results

To examine the NPC and nucleoplasmic distribution and expression of POM121 in human neuronal nuclei, control, and C9orf72 iPSNs were differentiated as previously described¹⁵. Postmortem human motor cortex and day 32 iPSNs were lysed and subjected to nuclei isolation and immunostaining as described above. NeuN positive isolated nuclei were imaged by super-resolution structured illumination microscopy (SIM) using a super-resolution structured illumination microscope (Zeiss) and processed using defau.......

Discussion

Given the recent identification of NCT deficits as an early and prominent phenomenon in multiple neurodegenerative diseases^{16,27,28,30,31}, there exists a critical need to thoroughly examine the mechanism by which this pathology occurs. As the NPC and its individual Nup proteins critically control functional NCT^39,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL conical tubes	Fisher Scientific	14-959-49A
Beckman Ultracentrifuge	Beckman Coulter
Cell Scrapers	Sarstedt	83.183
Collagen	Advanced Biomatrix	5005
Coverslips	MatTek	PCS-170-1818
Cytofunnel	Thermo Fisher Scientific	A78710020
Cytospin 4	Fisher Scientific	A78300003
Dounce Homogenizers	DWK Life Sciences	357542
DTT	Sigma Aldrich	D0632
Eppendorf tubes	Fisher Scientific	05-408-129
Goat Anti-Chicken Alexa 647	Thermo Fisher Scientific	A-21449
Goat Anti-Mouse Alexa 488	Thermo Fisher Scientific	A-11029
Goat Anti-Mouse Alexa 568	Thermo Fisher Scientific	A-11031
Goat Anti-Mouse Alexa 647	Thermo Fisher Scientific	A-21236
Goat Anti-Rabbit Alexa 488	Thermo Fisher Scientific	A-11034
Goat Anti-Rabbit Alexa 568	Thermo Fisher Scientific	A-11036
Goat Anti-Rabbit Alexa 647	Thermo Fisher Scientific	A-21245
Goat Anti-Rat Alexa 488	Thermo Fisher Scientific	A-11006
Goat Anti-Rat Alexa 568	Thermo Fisher Scientific	A-11077
Goat Anti-Rat Alexa 647	Thermo Fisher Scientific	A-21247
Hemacytometer	Fisher Scientific	267110
Microscope Slides	Fisher Scientific	12-550-15
Normal Goat Serum	Vector Labs	S-1000
Nuclei PURE Prep Nuclei Isolation Kit	Sigma Aldrich	NUC201	Contains Lysis Buffer, 10% Triton X-100, 2 M Sucrose Gradient, Sucrose Cushion Solution, and Nuclei Storage Buffer; Referenced in protocol as "nuclei isolation kit"
PBS	Thermo Fisher Scientific	10010023
PFA	Electron Microscopy Sciences	15714-S
Prolong Gold Antifade	Invitrogen	P36930	Referenced in protocol as "hard mount antifade mounting media"
SW 32 Ti Ultracentrifuge Rotor	Beckman Coulter	369694	Referenced in protocol as "ultracentrifuge rotor"
Triton X-100	Sigma Aldrich	T9284
Trypan Blue	Thermo Fisher Scientific	15-250-061
Ultracentrifuge Tubes	Beckman Coulter	344058
Nucleoporin Primary Antibodies			Primary antibodies suitable for immunofluorescent detection of invidual nucleoporins are available from multiple companies