Isolation of Pathology-Associated Intranuclear Inclusions

Abstract

An emerging theme in neurodegenerative diseases is the aggregation of proteins as inclusions in neural cells. Their presence is a useful tool in the differential diagnosis of the particular illness, although in no instance is the specific role of the inclusions in disease pathogenesis understood at present. However, apart from their role in the disease mechanism, the inclusions themselves may contain important molecular clues as to the mechanism(s) behind the specific inclusion-associated disease. Thus, isolation and analysis of the composition of the inclusions is likely to yield biochemical evidence of the cellular pathways that are involved in the disease process.

1 Introduction

Intracellular inclusions are common pathogenic hallmarks for a number of neurodegenerative diseases (1). For some disorders such as Huntington’s disease (Online Mendelian Inheritance in Man (OMIM)#143100), several of the spinocerebellar ataxias (SCAs), e.g., SCA3 (OMIM#109150), or fragile X-associated tremor/ataxia syndrome (FXTAS) (OMIM#300623), inclusions are found principally or exclusively in the nucleus. For other diseases, such as Parkinson disease (OMIM#168600) or multiple system atrophy (2), the associated inclusions are found in the cytoplasm. Although this chapter focuses on the isolation and purification of intranuclear inclusions, several of the principles involved with inclusion purification should be applicable to the isolation of inclusions in other locations.

Intranuclear inclusions/aggregates are typically ubiquitinated and are thought to accrete misfolded proteins due to the malfunction or overloading of the ATP-dependent ubiquitin proteasome system (3), although in most cases the relationship between the presence of ubiquitinated proteins in the inclusions and the pathogenic mechanism is unclear (4, 5). However, apart from being a distinguishing morphological feature of neurodegenerative diseases, inclusions may provide clues both to their own genesis as well as to the broader pathogenic mechanisms of the inclusion-associated disease.

To analyze the composition of the intranuclear inclusions associated with FXTAS, we have developed a two-phase protocol that rapidly yields highly purified intranuclear inclusions from postmortem brain cortical tissue. The first phase of the purification strategy, isolation of intact nuclei, serves several purposes. First, removal of the cytoplasm and cytoplasmic proteases reduces the rate of degradation of inclusion-associated proteins; second, in isolating inclusions from postmortem tissue, an intact nucleus provides a means of selection for cells that have not undergone extensive autolysis; third, the intact nuclear membrane provides some degree of protection against possible mechanical disruption during the initial phase of the purification.

The second phase of the protocol is the purification of inclusions from the isolated nuclei. The central strategy in this phase is the fractionation, by automated flow-based methods, of the inclusions based on their size and the presence of specific proteins (e.g., ubiquitin) that can be immunofluorescently tagged as a flow-selection marker. Purification using fluorescence-activated flow cytometric particle (inclusion) sorting is based on a strategy that is widely used for the separation of cells, based on specific surface markers. Similar strategies employing fluorescent staining have been used for the purification of Lewy bodies (6, 7), and polyglutamine aggregates from transfected mouse neuroblastoma cells (8). We have successfully applied this protocol to the purification of intranuclear inclusions from postmortem brain tissue of FXTAS patients and have applied several proteomic methods to characterize their protein complement (9).

2 Materials

2.1 Initial Characterization of Intranuclear Inclusions

• 1. SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA).

• 2. Phosphate-buffered saline containing Tween 20 (PBS-T): 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH 7.4, and 0.1% (v:v) Tween 20 (Sigma-Aldrich, St. Louis, MO, USA).

• 3. Antigen retrieval solution: 50 mM Tris-HCl, pH 9.5.

• 4. Parafilm “M” (Pechiney Plastic Packaging, Chicago, IL, USA).

• 5. Blocking solution: serum of the secondary antibody host species (e.g., rabbit, goat) diluted 1:10 (v/v) in PBS-T.

• 6. Fluorescent-labeled secondary antibodies: should be pre-absorbed against human proteins (assuming that the tissue of interest is of human origin). Jackson ImmunoResearch (West Grove, PA, USA) and Invitrogen (Molecular Probes, Carlsbad, CA, USA) supply secondary antibodies for staining and flow cytometry.

• 7. 4′,6-diamidino-2-(phenylindole) di-lactate (DAPI) (Sigma-Aldrich): 2 µM in H₂O.

• 8. Mounting and anti-fading solution: ProLong Gold (Invitrogen Molecular Probes).

• 9. Immunohistochemical staining kit: VECTASTAIN ABC system (Vector Laboratories, Burlingame, CA, USA).

2.2 Isolation of Nuclei from Tissue

• 1. Dounce homogenizer (15 mL) with loose pestle.

• 2. Homogenizing buffer (HB-Complete): 0.32 M sucrose, 50 mM Tris-HCl, 5 mM EDTA, 17 µg/mL of phenylmethanesulfonyl fluoride, pH 7.4, and 1 tablet/50 mL of Complete protease inhibitors (Roche, Indianapolis, IN, USA).

• 3. Nylon mesh (500 µm and 100 µm) (Small Parts Inc., Miami Lakes, FL, USA).

• 4. BC-Complete solution: 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 400 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N ′,N ′ tetraacetic acid (EGTA), with 1 tablet of Complete protease inhibitors (Roche)/50 mL.

2.3 Isolation of Crude Inclusions from Nuclei

• 1. BD-Complete solution: 40 mM Tris-HCl, 10 mM NaCl, 10 mM CaCl₂, 5 mM MgCl₂, pH 7.9, with 1 tablet of Complete protease inhibitors (Roche)/50 mL.

• 2. DNase I.

• 3. Nonidet P-40 (Sigma-Aldrich).

• 4. Dounce homogenizer (7 mL) with tightly fitting pestle.

2.4 Purification of Inclusions

• 1. Nylon mesh cell strainer, 40-µm pore size (BD Falcon, Bedford, MA, USA).

• 2. Fluorescent flow cytometer size reference beads of a size approximating that of the inclusions (PeakFlowTM; Invitrogen Molecular Probes).

• 3. Cell sorter: MoFlo (DakoCytomation, Fort Collins, CO, USA).

• 4. Cytocentrifuge: Shandon Cytospin (Thermo Scientific, Waltham, MA, USA).

3 Methods

3.1 Initial Characterization of Intranuclear Inclusions

Identification of at least two inclusion-associated proteins by immunostaining in situ is essential for the success of the current inclusion isolation protocol. Many inclusions stain positively for the presence of ubiquitin, a posttranslational modification associated with proteins destined for degradation or translocation within the cell. However, given the widespread distribution of ubiquitin, this is not sufficiently specific as a fractionation tag for inclusions. Therefore, it is necessary to identify at least one additional inclusion protein, colocalizing in a specific manner with the ubiquitin-positive inclusions, to provide adequate specificity of the separation method. Because many disease-associated inclusions likely contain misfolded proteins, a probable candidate for a second protein tag is one of the chaperone proteins (e.g., small or large heat shock-related proteins (HSPs)). Colocalization of protein candidates in the inclusions must be established by immunofluorescent staining. Additionally, immunofluorescent staining should be confirmed by use of chromogen substrates, because particle autofluorescence can be misleading in the interpretation of fluorescence staining. Further important information that can be gleaned from these initial studies includes the size of the inclusions, the fraction of nuclei that contain inclusions, and how much starting material is necessary to yield sufficient material for downstream analysis.

• 1. Prepare formalin-fixed, paraffin-embedded tissue sections (~5 µm) on SuperFrost plus slides for in situ staining (see Note 1).

• 2. Deparaffinize and rehydrate the sections by heating the slides at 60°C for 10–20 min.

• 3. Transfer the slides immediately to xylene and incubate at room temperature for 3–5 min.

• 4. Transfer the slides to 100% ethanol for 5 min, repeat once, then to 95% ethanol for 5 min, repeat once, then to 90% ethanol for 3 min, to 85% ethanol for 3 min, and then to PBS-T.

• 5. To perform antigen retrieval, submerge a Coplin-style jar containing antigen retrieval solution in a water bath at room temperature and heat the water bath to ~95°C.

• 6. Place the slides in the jar, cover with a loose-fitting lid, and place the jar in the water bath for 20–30 min. Carefully remove the jar from the water bath and allow the slides to cool gradually in the jar.

• 7. Transfer the slides to PBS-T and wash them by exchanging the PBS-T solution, repeat for a total of three washes.

• 8. Block the slides against nonspecific binding of secondary antibodies. Line a tray with a strip of Parafilm “M.” Carefully place ~250–300 µL of blocking solution on the Parafilm. Flip the slide over, tissue side down, onto the blocking solution. Repeat for the remaining slides and incubate them at room temperature for 1–2 h.

• 9. Incubate the slides with primary antibodies, diluted in blocking solution according to the vendor’s specifications. For immunofluorescent staining, the slides can be incubated with both antibodies together, but these must be generated in different host species. For immunocytochemistry with chromogen substrates, the antibodies must be evaluated individually (see Note 2). Flip the slides over, as for the blocking step, onto an aliquot of primary antibodies. Incubation times and temperatures depend on the affinity and specificity of the antibodies; generally, overnight incubation at 4°C is sufficient.

• 10. Wash the slides in PBS-T for 5 min, repeat four times more.

• 11. Incubate the slides for 1–2 h with labeled secondary antibodies diluted in blocking solution. Generally, dilutions of 1/1,000 (v/v) for fluorescent staining and 1/200 for chromogen staining are adequate. Again, for fluorescent staining the fluorophore-labeled secondary antibodies can be combined (see Note 3).

• 12. Wash the slides in PBS-T for 5 min, repeat four times more.

• 13. For fluorescence microscopy, counterstain the slides in DAPI solution for 5 min. Place a drop of ProLong mounting media on the slides and then a coverslip.

• 14. For chromogen staining, ready-to-use kits such as the VECTASTAIN ABC system are convenient and provide good-quality staining for immunocytochemistry. Counterstain the slides with hematoxylin and add a coverslip with a compatible mounting medium such as Crystal/Mount (Biomeda, Foster City, CA, USA).

• 15. Evaluate microscopically the specificity of the staining and the colocalization of the antibodies on inclusions, the distribution of inclusions in the tissue, and the size of inclusions. Minimally, a pair of antibodies (Ab I and Ab II) with high specificity for inclusion-associated proteins (protein I and protein II) must be established for subsequent isolation and purification of the inclusions.

3.2 Isolation of Nuclei from Tissue

The separation of nuclei from tissue can, in principle, be accomplished through one of several methods. However, we found that methods employing isopycnic banding in iso-osmotic media were not useful for the efficient isolation of inclusion-bearing nuclei, for reasons that were not explored further. Gentle methods of tissue homogenization prior to the isolation of intact nuclei generally yield acceptable quantities of intact inclusions; however, harsher disruption methods such as sonication or motorized homogenizers appear to fracture the inclusions into smaller aggregates and significantly complicate their further purification.

• 1. Cut ~1–2 g of tissue into ~1 mm³ pieces and suspend in four volumes of HB-Complete (see Note 4).

• 2. Homogenize the tissue on ice in a Dounce homogenizer (see Note 5) fitted with a loose pestle. Ten to 15 downward strokes are usually adequate.

• 3. Filter the homogenate successively through 500-µm and 100-µm nylon mesh. Rinse the homogenizer and the mesh with 2 mL of HB-Complete and pool the wash with the homogenate.

• 4. Centrifuge the filtered homogenate at 1,500×g for 10 min at 4°C.

• 5. Wash the pelleted nuclei 3× in HB-Complete and resuspend the pellet in BC-Complete such that final volume is 1.5–2.0 mL.

• 6. Make slides with nuclear smears for future staining experiments: apply ~1–2 µL of resuspended nuclei to a SuperFrost Plus slide and smear across the surface using another slide. Prepare 20–40 slides and fix them in 70% v/v methanol for 15 min, allow to dry, and store at −20°C.

3.3 Isolation of Crude Inclusions from Nuclei

The inclusions are released by disruption of the nuclear membrane and are easily pelleted from the resulting solution. However, despite several washes the inclusions may remain associated with DNA, making them prone to clumping and difficult to purify. A step involving mild DNase digestion removes extrinsic DNA, although a caveat with such treatment is that it may also affect proteins that are loosely associated with the inclusions.

• 1. To the suspension of nuclei, add Nonidet P-40 to a final concentration of 0.25% v/v (see Note 6).

• 2. Rotate the suspension on an end-over-end tube mixer for 15 min at 4°C.

• 3. Transfer the nuclei to a Dounce homogenizer (see Note 7) and homogenize on ice with 8–12 downward strokes of a tightly fitting pestle.

• 4. Pellet the crude inclusions by centrifuging at 2,000×g for 5 min at 5°C.

• 5. Wash the pellet 2× in BC-Complete and resuspend in 200–500 µL of BD-Complete.

• 6. Add DNase I to a final concentration of 500 U/mL and incubate on a rotator at 20°C for 30 min.

• 7. Centrifuge at 2,000×g for 5 min at 5°C.

• 8. Resuspend the pellet of crude inclusions in BC-Complete using 1.0 mL of BC-Complete per gram of original tissue, aliquot, and store at −80°C.

3.4 Purification of Inclusions

The second phase of the inclusion purification process uses fluorescence-activated flow cytometric particle sorting. As noted in the Introduction, similar strategies have been applied to the separation of cells based on fluorescent tagging of cell surface markers, and for the purification of Lewy bodies and polyglutamine inclusions. Based on estimates of the range of inclusion diameters (for particle size fractionation) and immunospecific fluorescent staining, inclusions can be preparatively sorted and collected. Visual examination by immunofluorescence microscopy is necessary to confirm that the sorting has been successful and that the morphology of the inclusions has not substantially changed during the purification process.

• 1. Pellet the crude inclusions by centrifugation at 2,000×g for 5 min at 5°C.

• 2. Prepare an appropriate blocking solution containing 5% v/v serum of the secondary antibody host species in PBS-T. Resuspend the crude inclusions in 500 µL of this blocking solution.

• 3. Incubate with rotation at 4°C for 2 h (see Note 8).

• 4. Pellet the inclusions at 2,000×g for 5 min at 4°C.

• 5. Resuspend the crude inclusions in between 200 and 400 µL of the appropriate primary antibody diluted in blocking buffer. Initially, antibody combinations and controls must be run to establish gates for sorting the inclusions (Table 13.1).

• 6. Rotate the crude inclusions overnight at 4°C and spin at 2,000×g to pellet them.

• 7. Resuspend the pelleted inclusions in ~1.0 mL of PBS-T and spin at 2,000×g to pellet them. Repeat four times more.

• 8. Prepare the secondary antibodies and incubate the pelleted inclusions in between 200 and 400 µL of the secondary antibody solutions for 2 h at 4°C with rotation; protect from light.

• 9. Centrifuge the inclusions at 2,000×g, resuspend the pellet in ~1.0 mL of PBS-T, spin at 2,000×g to pellet, and repeat four times more.

• 10. Resuspend the inclusions in ~1.0 mL of PBS-T and pass through a 40-µm-pore size nylon mesh cell strainer sieve. Wash the tube and sieve with a second aliquot of PBS-T and pool the wash with the sieved inclusions. Keep the samples on ice for sorting.

• 11. Fit the MoFlo cell sorter with a 70-µm nozzle tip.

• 12. Determine the excitation wavelengths and detection filters (bandpass) for fluorescent antibody probe I and fluorescent antibody probe II.

• 13. Estimate the size and complexity of the inclusions by assessing forward and side scatter of unlabeled inclusions (see Note 9).

• 14. Set the initial gate to exclude very small particles (<1.0 µm) and large debris (>10 µm).

• 15. Examine unlabeled inclusions and determine their distribution on each axis (forward and side scatter).

• 16. Examine inclusions incubated with Ab I and fluorescent probe I.

• 17. Examine inclusions incubated with Ab II and fluorescent probe II.

• 18. Set compensation for detection of signals from both fluorescent probe I and fluorescent probe II.

• 19. Examine inclusions incubated with fluorescently labeled secondary antibodies in the absence of primary antibodies (see Note 10).

• 20. Examine inclusions labeled with both antibodies and both probes, set gates and collect particles that are highly positive for both fluorescent probes (see Note 11). Sort the particles at ~19,000 events/sec with a coincidence rate at or below 9%.

• 21. Re-examine a small sample of the sorted population that is double positive for fluorescent probes I and II, using intensity versus forward scatter to verify positive staining (see Note 12).

• 22. Collect double-positive particles in PBS-T Complete at 4°C.

• 23. Dilute a small sample of the double-positive inclusions/particles ~1:20 in PBS-T, and cytospin onto slides for microscopic evaluation. Purification of FXTAS-related inclusions by flow cytometry is shown in Fig. 13.1 and immunofluo-rescence staining at various steps of their purification in Fig. 13.2.

Table 13.1 Antibody controls essential for flow cytometry

Fig. 13.1

Purification of FXTAS-related intranuclear inclusions (INC) by flow cytometry. a Forward scatter (FSC) versus side scatter (SSC) assessment of unlabeled crude inclusions (no primary or secondary antibodies) for the determination of size gating. b Secondary antibodies only, FSC versus fluorescence. c Immunolabeled with rabbit anti-ubiquitin and Alexa 488 goat anti-rabbit. d Immunolabeled with mouse anti-αB-crystallin and Alexa 594 goat anti-mouse IgG. e Immunolabeled with rabbit anti-ubiquitin/Alexa 488 goat anti-rabbit IgG, and mouse anti-αB-crystallin/Alexa 594 goat anti-mouse IgG

Fig. 13.2

Immunofluorescence staining of inclusions at various steps in their isolation and purification. FXTAS-related inclusions were processed for immunostaining with polyclonal rabbit antibody to ubiquitin and mouse monoclonal antibody to αB-crystallin. Secondary antibodies (Alexa 488 goat anti-rabbit and Alexa 555 goat anti-mouse) were visualized by fluorescence microscopy and nuclei were counterstained with DAPI. Merged images demonstrate the presence of both ubiquitin and crystallin in the inclusions. Purified nuclei contain ubiquitin- and crystallin-positive inclusions; no staining of the inclusions was observed with antibodies to nucleolar-related proteins. Staining of nonsorted, crude inclusions demonstrates the additive specificity of the two antibodies and improves the identification of the inclusions in the pool of nuclear material. Immunofluorescent staining of inclusions is an important tool in monitoring the progress of the isolation and purification process. To view this figure in color, see COLOR PLATE 4

4 Notes

• 1. Specimens from various regions of the tissue of interest will provide information on the distribution of inclusions, particularly the best region for the collection of the greatest numbers of inclusions.

• 2. Antibody controls must be tested, including non- or pre-immune sera and secondary antibodies without primary antibodies.

• 3. Use fluorophores with minimal overlap of excitation/emission wavelengths.

• 4. Control, non-inclusion bearing tissue should be processed in the same fashion to confirm the specificity of the isolation protocol.

• 5. A 15-mL-size homogenizer nicely handles the resuspension and the displacement of the tissue sample during the homogenizing process.

• 6. Because NP-40 is very viscous, an intermediate dilution (10% v/v) in BC-Complete allows for more accurate addition to achieve the desired final concentration.

• 7. We recommend a 7-mL-size homogenizer for this step.

• 8. Following this incubation, it is advisable to plan ahead as to how many antibody permutations must be evaluated. Prepare and parcel aliquots according to these conditions and proceed to the next step.

• 9. Size approximation is based on fluorescent reference beads. The size of the beads should approximate the size of the inclusions.

• 10. These inclusions should appear similar to unlabeled crude inclusions. If strong fluorescence is observed for either probe in the absence of primary antibody, more rigorous washing or further dilution of the secondary antibody may be necessary to eliminate nonspecific binding.

• 11. An intermediate population, slightly less positive for both fluorescent probes but likely far more abundant, can be collected and is useful for testing downstream analytical/proteomic methods.

• 12. The intensity of fluorescence may be reduced due to bleaching from the first round of sorting.