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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 understood2,3,4,5,6,7,8,9,10,11,12. Recently, a G4C2 hexanucleotide repeat expansion in the first intron of the C9orf72 gene was identified as the most common genetic cause of the related neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD)13,14. Several studies now support a central role for disruptions in the nuclear transport machinery, including nuclear pore complexes (NPCs) and nuclear transport receptors (NTRs, karyopherins), as being causative of C9orf72 ALS15,16. In non-dividing cells within the rat brain, scaffold nucleoporins (Nups) are extremely long-lived. As a result, alterations in NPCs and NCT have been reported during aging17,18,19,20. Moreover, some nucleoporins or transportins, when mutated, are linked to specific neurological diseases21,22. For example, mutations in Nup62 have been linked to Infantile Bilateral Striatal Necrosis (IBSN), a neurological disorder affecting the caudate nucleus and putamen23; mutations in Gle1 have been implicated in the fetal motor neuron disease Human Lethal Congenital Contracture Syndrome-1 (LCCS1)24; and mutations in Aladin are causative of Triple-A Syndrome25. Alterations in functional NCT are exacerbated in age-related neurodegenerative diseases such as ALS, Huntington's Disease (HD), and Alzheimer's Disease (AD)16,26,27,28,29,30,31. In addition, specific Nups and NTRs have been reported as modifiers of C9orf72 mediated toxicity in the Drosophila eye28 or biochemically modify the aggregation state of disease-linked proteins such as FUS and tau27,32,33,34. Collectively, these early studies suggest that altered NCT may be a primary and early pathological feature of ALS and FTD. Studies in overexpression-based model system have suggested that mislocalization of specific Nups and karyopherins may impact NCT16,35,36,37,38. However, these pathology studies do not actually link cytoplasmic accumulations of NPC proteins to defects in the structure or function of the NPC. For example, this pathology may simply reflect the dysregulation of cytoplasmic pools of Nup proteins with little impact on NPC composition and function. In contrast, a recent study employing super resolution structured illumination microscopy (SIM) demonstrates the emergence of a significant injury to the NPC itself characterized by reduction in specific Nup levels within the nucleoplasm and NPCs of human C9orf72 ALS/FTD neurons ultimately leading to altered NPC function as an early initiating event in pathogenic disease cascades15.

The passage of macromolecules between the nucleus and cytoplasm is governed by the nuclear pore complex (NPC). The NPC is a large macromolecular complex embedded in the nuclear envelope comprised of multiple copies of 30 nucleoporin proteins (Nups)39,40,41. Although Nup stoichiometry varies among cell types42,43,44, maintenance of overall NPC composition is critical for NCT, genome organization, and overall cellular viability39,41,45,46. As a result, altered NPC composition and subsequent defects in functional transport are likely to impact a myriad of downstream cellular functions. The Nup constituents of the NPC are highly organized into multiple subcomplexes, including the cytoplasmic ring and filaments, central channel, outer ring, inner ring, transmembrane ring, and nuclear basket. Collectively, scaffold Nups of the inner, outer, and transmembrane rings anchor NPCs within the nuclear envelope and provide anchor points for Nups of the cytoplasmic ring, central channel, and nuclear basket. While small molecules (<40-60 kD) can passively diffuse through the NPC, the active transport of larger cargoes is facilitated by interactions between nuclear transport receptors (NTRs, karyopherins) and the FG Nups of the cytoplasmic filaments, central channel, and nuclear basket39,40,41,45. Also, a handful of Nups can additionally function outside of the NPC, within the nucleoplasm, to regulate gene expression46,47.

Given that the lateral dimension of a single human NPC is approximately 100-120 nm40, standard widefield or confocal microscopy is insufficient to resolve individual NPCs48. Electron microscopy (EM) techniques such as TEM or SEM are often used to evaluate the overall structure of NPCs39,40. Despite the advantages of these techniques for resolving NPC ultrastructure, they are less commonly used to evaluate the presence of individual Nup proteins within the NPC. The technical limitations of combining antibody or tag-based labeling with these state-of-the-art technologies, TEM and SEM, do not always allow for an accurate and reliable assessment of individual Nups themselves within NPCs or the nucleoplasm. Further, these techniques can be technically challenging and are not yet widely accessible to all researchers. However, recent advances in light and fluorescence microscopy have increased the accessibility of super-resolution imaging technologies. Specifically, SIM affords the unparalleled opportunity to image individual Nups with a resolution that approaches the lateral dimensions of one human NPC40,48,49,50,51. In contrast to other super-resolution approaches such as stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED), SIM is compatible with conventional antibody-based immunostaining49. Thus, SIM allows for a comprehensive analysis of all Nups for which a specific Nup antibody is available. The ability to sample and image multiple different Nups in the same preparation provides significant advantages to other imaging methods when surveying the many proteins that comprise the NPC. The following procedure details an optimized protocol for evaluating individual Nup components of the NPC using nuclei isolated from induced pluripotent stem cell (iPSC) derived neurons (iPSNs) and postmortem human central nervous system (CNS) tissues.

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 ΞΌL 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 for 5 min.
  3. Aspirate the collagen solution and let the slides air dry at room temperature for about 1 h.

2. Preparation of lysis buffer and sucrose gradients

  1. Prepare the lysis buffer and sucrose gradient solutions according to the nuclei isolation kit protocol.
    1. For each sample, prepare a 50 mL conical tube of lysis buffer by combining 11 mL of the supplied lysis buffer, 110 ΞΌL of the supplied 10% Triton X-100 solution, and 11 ΞΌL of freshly prepared or thawed 1 M DTT (Dithiothreitol).
    2. For each sample, use a 50 mL conical tube to prepare a 1.85 M sucrose cushion solution by combining 27.75 mL of the supplied 2 M sucrose cushion solution, 2.25 mL of the supplied sucrose cushion buffer, and 30 ΞΌL of freshly prepared or thawed 1 M DTT.
      NOTE: A 1.85 M sucrose cushion solution is optimal for iPSNs and postmortem human CNS tissue, but adjust it for other cell types or tissue samples. Additional details for HEK293 cells, iPSC-derived astrocytes, and enrichment of oligodendrocyte nuclei from postmortem CNS tissues have recently been published15.
  2. Gently mix lysis buffer and sucrose cushion solutions by inverting conical tubes and store it on ice.

3. Lysis of iPSNs and postmortem human CNS tissue

  1. Before proceeding with the lysis, place the ultracentrifuge rotor (without sample holders) in the ultracentrifuge and allow it to pre-cool to 4 Β°C.
  2. Lysis of iPSNs.
    NOTE: Lysis protocols differ for iPSNs and postmortem human CNS tissue. Follow the protocol for the sample type specified below (step 3.2: iPSNs, step 3.3: postmortem human CNS tissue).
    1. Remove the iPSNs from the incubator and aspirate the media. Rinse briefly with 1x PBS and add the lysis buffer directly to the well or plate.
      NOTE: Take care to adequately rinse iPSNs with a sufficient quantity of 1x PBS to remove debris and dead cells. The volume of lysis buffer to be added to iPSN plates will vary. Please refer to Table 1. Ensure that the starting material is >2.5 million iPSNs.
    2. Scrape the iPSNs with a cell scraper and transfer them to the remaining lysis buffer in the 50 mL conical tube.
    3. Cap each conical tube and vortex the samples for 20 s to facilitate iPSN lysis.
    4. Let the samples sit on ice for 1-2 min before proceeding.
  3. Lysis of postmortem human CNS tissues.
    1. Weigh out ~100 mg of frozen postmortem human CNS tissue by cutting with a razor blade in a Petri dish placed on a bed of dry ice. Be sure to work on a surface surrounded by disposable pads to avoid local tissue contamination. Pay attention to specific gray matter versus white matter dissections (e.g., cortical mantel versus underlying white matter) visible at the time of tissue dissection.
      NOTE: Wear the appropriate PPE, including eye protective wear and gloves when handling frozen postmortem human tissue samples. This step can also be completed ahead of time and tissue aliquots stored at -80 Β°C. 50 mg is a sufficient quantity for nuclei isolation. However, more than 100 mg tissue often yields incomplete isolation of nuclei, as evidenced by the presence of intact cytoplasm surrounding some nuclei.
    2. Prepare the Dounce homogenizer by cleaning and thoroughly rinsing with distilled water. Chill the homogenizer by placing it on ice.
    3. Add 100 mg of tissue to freshly cleaned, rinsed, and chilled (on ice) Dounce homogenizer containing 2 mL of the prepared lysis buffer.
    4. Homogenize in Dounce homogenizer using the standard procedure on ice.
      NOTE: Typically, 10-20 strokes per pestle is sufficient to move through the sample without resistance.
    5. Transfer 2 mL of postmortem human brain homogenate to the remaining 9 mL of lysis buffer in conical tubes.
    6. Vortex vigorously for 30 s and let it sit on ice for 5 min to facilitate lysis.

4. Isolation of nuclei from iPSNs and postmortem human CNS tissue

  1. Layer 10 mL of 1.85 M sucrose cushion solution (made in step 2.1.2) at the bottom of an ultracentrifuge tube and add 18 mL of 1.85 M sucrose cushion solution to each lysate.
  2. Gently mix the lysate/sucrose cushion solution (combined in step 4.1) by inverting the 50 mL conical tube.
  3. Slowly add 28 mL of lysate/sucrose cushion solution mix to the top of the 10 mL of 1.85 M sucrose cushion solution in the ultracentrifuge tube (from step 4.1).
    NOTE: There is a total of 29 mL lysate/sucrose cushion solution mix in the 50 mL conical tube (steps 4.1-4.2). Leave 1 mL of this lysate/sucrose cushion solution mix in the 50 mL conical tube. This ensures accurate and consistent pipetting among all samples due to the viscosity of the sucrose cushion solution.
  4. Place the ultracentrifuge tubes in the holders and add an additional 1.85 M sucrose cushion solution (made in step 2.1.2) as needed to balance the samples.
  5. Place the sample holders in the rotor in the chilled ultracentrifuge and spin at 30,000 x g, 4 Β°C for 45 min.
  6. Remove the ultracentrifuge tubes from the sample holders and discard the supernatants. Nuclei will be visible as pellets on the bottom sides of the ultracentrifuge tube.
  7. Resuspend the nuclei pellets in 1 mL of the supplied nuclei storage buffer by vortexing and transfer to a microcentrifuge tube.
  8. Centrifuge the microcentrifuge tubes at 2,500 x g, 4 Β°C for 5 min.
  9. Remove the supernatant and resuspend the nuclei by vortexing in fresh 1 mL of the supplied nuclei storage buffer.
  10. Proceed with immunostaining or store the nuclei at -80 Β°C.
    ​NOTE: Nuclei can be stored at -80 Β°C for up to 6 months and be subjected to two freeze/thaw cycles before structural integrity is compromised.

5. Immunostaining of the isolated nuclei

  1. Take a 10 ΞΌL aliquot of the nuclei suspension and count using a hemacytometer or an automated cell counter.
  2. Assemble the prepared slides (from step 1.3) into the cytofunnels.
  3. To each cytofunnel, layer 200 ΞΌL of fresh nuclei storage buffer and ~100,000 nuclei.
  4. Gently spin the nuclei onto the slides by placing the cytofunnels into a cytospin and centrifuging for 3 min at 100 x g.
  5. Unclip the cytofunnels and immediately add ~100 ΞΌL of 4% PFA (paraformaldehyde) to the slides and incubate for 15 min at room temperature.
    NOTE: Nuclei can also be fixed with methanol. Use fixation method appropriate for each antibody. If using methanol, skip the permeabilization step (5.7). Cytofunnels are single-use; discard them at this point.
  6. Wash the nuclei 3x for 5 min with 1x PBS.
  7. Permeabilize the nuclei with 0.1% Triton X-100 in 1x PBS for 10 min at room temperature.
  8. Block the nuclei in block solution (10% goat or donkey serum in 1x PBS) for 30 min at room temperature.
  9. Incubate the nuclei with primary antibodies diluted in block solution overnight at 4 Β°C.
  10. Wash the nuclei 3x for 5 min with 1x PBS.
  11. Incubate the nuclei with secondary antibodies diluted in block solution for 1 h at room temperature.
    NOTE: Alexa Fluor 488, 568, and 647 secondary antibodies are preferred.
  12. Wash the nuclei 3x for 5 min with 1x PBS.
  13. OPTIONAL: Incubate the nuclei for 5 min with DAPI or Hoechst followed by two additional 5 min washes with 1x PBS.
  14. Hold a lint-free wipe at the edge of the circle containing nuclei to remove the last PBS wash completely.
  15. Add 1 drop (~10 ΞΌL) of a hard mount antifade mounting media (without DAPI) to each slide and gently place high tolerance 18 mm x 18 mm square coverslips on each slide.
    NOTE: When using a hard-mount media, slide sealing is not necessary if slides are imaged within an appropriate time frame. Nup immunoreactivity has typically remained stable on unsealed slides stored at 4 Β°C for ~6 months. However, the edges of the coverslip can be sealed with a thin layer of nail polish if a wet-mount media is used or for prolonged storage.
  16. Keep the slides protected from light and let them cure overnight at room temperature.
  17. Image the nuclei can by super-resolution structured illumination microscopy (SIM).
    NOTE: Zeiss, Nikon, and GE Healthcare all manufacture SIM microscopes. Follow the system-specific protocols for image acquisition and processing. Use imaging parameters (laser power, filter sets, exposure time) appropriate for each Nup and fluorophore. When imaging, avoid edges of the circle (from step 1.1) as these nuclei are typically flattened and spread out as a result of the centrifugation step (5.4). Fully deconvolved and processed images can be subjected to a number of analyses, including spot detection and volume measurements using standard 3D analysis modules in FIJI or Imaris image analytics software. Additional details on analysis methods have been recently published15.

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 diseases16,27,28,30,31, 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 NCT39,

Disclosures

The authors declare no competing financial interests.

Acknowledgements

Postmortem human CNS tissues were provided by the Johns Hopkins ALS Autopsy Bank and the Target ALS Postmortem Tissue Core. This work was supported by the ALSA Milton Safenowitz Postdoctoral Fellowship (ANC), as well as funding from NIH-NINDS, Department of Defense, ALS Association, Muscular Dystrophy Association, F Prime, The Robert Packard Center for ALS Research Answer ALS Program, and the Chan Zuckerberg Initiative.

Materials

NameCompanyCatalog NumberComments
50 mL conical tubesFisher Scientific14-959-49A
Beckman UltracentrifugeBeckman Coulter
Cell ScrapersSarstedt83.183
CollagenAdvanced Biomatrix5005
CoverslipsMatTekPCS-170-1818
CytofunnelThermo Fisher ScientificA78710020
Cytospin 4Fisher ScientificA78300003
Dounce HomogenizersDWK Life Sciences357542
DTTSigma AldrichD0632
Eppendorf tubesFisher Scientific05-408-129
Goat Anti-Chicken Alexa 647Thermo Fisher ScientificA-21449
Goat Anti-Mouse Alexa 488Thermo Fisher ScientificA-11029
Goat Anti-Mouse Alexa 568Thermo Fisher ScientificA-11031
Goat Anti-Mouse Alexa 647Thermo Fisher ScientificA-21236
Goat Anti-Rabbit Alexa 488Thermo Fisher ScientificA-11034
Goat Anti-Rabbit Alexa 568Thermo Fisher ScientificA-11036
Goat Anti-Rabbit Alexa 647Thermo Fisher ScientificA-21245
Goat Anti-Rat Alexa 488Thermo Fisher ScientificA-11006
Goat Anti-Rat Alexa 568Thermo Fisher ScientificA-11077
Goat Anti-Rat Alexa 647Thermo Fisher ScientificA-21247
HemacytometerFisher Scientific267110
Microscope SlidesFisher Scientific12-550-15
Normal Goat SerumVector LabsS-1000
Nuclei PURE Prep Nuclei Isolation KitSigma AldrichNUC201Contains Lysis Buffer, 10% Triton X-100, 2 M Sucrose Gradient, Sucrose Cushion Solution, and Nuclei Storage Buffer; Referenced in protocol as "nuclei isolation kit"
PBSThermo Fisher Scientific10010023
PFAElectron Microscopy Sciences15714-S
Prolong Gold AntifadeInvitrogenP36930Referenced in protocol as "hard mount antifade mounting media"
SW 32 Ti Ultracentrifuge RotorBeckman Coulter369694Referenced in protocol as "ultracentrifuge rotor"
Triton X-100Sigma AldrichT9284
Trypan BlueThermo Fisher Scientific15-250-061
Ultracentrifuge TubesBeckman Coulter344058
Nucleoporin Primary AntibodiesPrimary antibodies suitable for immunofluorescent detection of invidual nucleoporins are available from multiple companiesΒ 

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