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 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
Representative Results
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,<sup class="…
Declarações
The authors have nothing to disclose.
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
| 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 |
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