An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Podsumowanie

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

Streszczenie

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

Wprowadzenie

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a fatal demyelinating disease resulting from loss of function mutations in the galatocerebrosidase (galc) gene¹. The most prevalent form of GLD is the infantile variant which is typified by onset in early childhood and characterized by an aggressive clinical course of motor and cognitive decline leading to premature death often before five years of age^2,3. Genetic testing is used to verify a diagnosis of GLD⁴. Neuropathology of GLD reveals widespread demyelination, neuronal atrophy, astrogliosis and presence of engorged multi-nucleated microglia called globoid cells^5-7. The identification of globoid cells, often containing tubulous inclusions in their cytoplasm, has been a defining feature of GLD for the past 97 years, although the specific function of these conspicuous cells has remained elusive.

The involvement of non-myelinating glia (microglia and astrocytes) in the pathogenesis of GLD has long been considered a secondary response to the profound demyelination in this disease⁸. Interestingly, the first description of this disease, made by Knud Krabbe in 1916⁵, reported formation of multinucleated phagocytes containing lipid debris that have been named 'globoid cells' and are a defining characteristic of this disease.

Globoid cells are the hallmark feature of GLD pathology, although their role in GLD has long been ignored. Interestingly, these cells are among the earliest characteristic changes in CNS tissue of GLD. This lack of knowledge may have been due to the assumption that the formation of multinucleated phagocytes, called giant cells in other diseases, are typically considered as a consequence of pathology rather than an initial pathogenic driving force⁹. Therefore, there have been few studies investigating the mechanism by which globoid cells are formed from phagocytes, particularly in the CNS of GLD. The procedure described in this report focuses on the importance of globoid cell formation in the CNS and our previous demonstration that psychosine-induced multinucleation of microglia in vitro and these cells exhibited higher levels of phagocytic activity. Consistent with these observations, globoid cells in twitcher brains frequently contain PAS-positive debris, suggesting high levels of phagocytic activity. Globoid cells are also found to be immunopositive for ferritin (a microglia marker)¹⁰, KP-1/CD68 (a monocyte marker), and some are also positive for vimentin (an intermediate filament protein and marker of astrocytes and activated microglia)¹¹, HLA-DRa (an MHCII surface receptor), and TNF-α⁷, and Iba-1 (a calcium binding protein used to identify microglia)¹². Based on this collection of markers, globoid cells originate from microglia that develop a unique phenotype.

Despite their uniqueness, the specific function and contribution of GCs to GLD pathogenesis has been largely overlooked. Globoid cells have been thought to be a secondary consequence of chronic demyelination. However, past studies examining the temporal association of globoid cells to the white matter pathology of GLD have identified the presence of globoid cells in the late embryonic to early postnatal periods; times preceding oligodendrocyte apoptosis and overt demyelination¹³. Thus, the temporal sequence of development of the neuropathology in GLD suggests that globoid cells are formed in advance of demyelination in this disease¹⁴. This led to our hypothesis that the early formation of globoid cells in GLD may represent a defining pathogenic event rather than a secondary, reactive response to oligodendrocyte damage¹⁵. Additionally, dysregulation of microglial activity in GLD has been considered a factor limiting the long-term efficacy of hematopeotic stem cell therapies for treating this disease¹⁶. Thus, investigating the cellular functions and regulation of microglia, and globoid cells, in response to psychosine is expected to provide new insights in the pathogenesis of GLD.

Until recently, the lack of an appropriate model in which to study globoid cell formation had limited the understanding of the precise function and contribution of these cells to the pathology of GLD. In recent studies, it was determined that globoid-like cells can be formed in direct response to psychosine, a pathogenic lipid toxin that accumulates in GLD. We found that microglia, but not macrophages, are activated and transformed into globoid cells in primary glial cultures in response to psychosine¹⁵. This transformation into globoid cells was found to be mediated by the extracellular protease, matrix metalloproteinase (MMP)-3¹⁵. More recently, we have extended these findings and determined that psychosine-activated microglia and globoid cells developed in this in vitro model system are potently toxic to oligodendrocytes and oligodendrocyte progenitor cells. Hence, when considered in the context of GLD, the early accumulation of psychosine and formation of globoid cells prior to demyelination would support an emerging primary and possibly pathogenic role for microglia in this disease.

We propose that study of globoid cell formation will reveal new information about the pathogenesis of GLD that will contribute to our understanding of this disease. Moreover, this new cellular model of GLD may provide a new format from which novel therapeutic approaches to address pathological changes in this disease could be tested. Hence, in this report we provide a detailed protocol for the in vitro development of psychosine-induced globoid cells from primary cultures of non-myelinating glia.

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Protokół

All procedures involving animals were performed in accordance with the Policy on Humane Care and Use of Laboratory Animals set forth by the Office of Laboratory Animal Welfare (NIH) and only with approval from the Institutional Animal Care and Use Committee (IACUC) of the University of Connecticut Health Center.

1. Preparation of Mixed Glial Cultures

1. Sterilize all instruments prior to use. Add 3 ml of ice-cold sterile Hank’s balanced salt solution (HBSS) containing no cations (Mg²⁺ or Ca²⁺) to three sterile 60 mm Petri dishes maintained on ice to keep cold.
2. Isolate the cortices from postnatal P0-P2 mouse pups, as previously described^17,18.
   NOTE: Subcortical structures, such as hippocampi, are not included in these cultures.
3. Carefully remove the meninges and then transfer the isolated cortices to fresh HBSS and dissociate the tissues enzymatically, using a neural tissue dissection kit according to the manufacturer's protocol.
4. Plate the cells into sterile T-175 flasks and incubate overnight in media [Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) containing 1x penicillin/streptomycin].
5. Replace all media the following day with fresh media. Continue to incubate cultures, with regular media changes, for approximately 3 weeks or until a cellular monolayer is established.
   NOTE: This monolayer will be comprised of astrocytes and microglia with an approximately 10:1 ratio¹⁹.

2. Globoid Cell Induction in Mixed Glial Cultures

1. Prepare the ECM-coated coverslips.
   1. Soak circular coverslips with sterile 1 N hydrochloric acid (HCl) in a sterile Petri dish for 30 min. Wash coverslips with sterile water 3x and then soak in 95% EtOH for at least 20 min. and then let air-dry.
   2. Place droplets of diluted extracellular matrix (ECM) protein coating materials onto a sheet of Parafilm (e.g. 150-250 µl/droplet of laminin 10 μg/ml dissolved in sterile phosphate buffer solution [PBS]). Distribute the droplets so that coverslips will not touch when placed onto these droplets. Gently place each dried coverslip onto a droplet using forceps.
   3. Leave the coverslips on the droplets for approx. 4-6 hr inside the culture hood with the sash closed.
   4. Place the ECM-coated coverslips (ECM-coated side up) into each of the wells of a 24-well plate. Wash each well with sterile PBS 3x and keep at 4 °C until needed.
2. Preparing Glial Cells for Plating onto Coverslips.
   1. Aspirate the media from the glial cultures and gently wash the glial monolayer 3x with 10 ml of sterile PBS. Next, add 8-10 ml trypsin-EDTA solution to the T-175, and incubate for 5-10 min at 37 °C.
   2. When most of the monolayer has been detached, add 20 ml of complete media to the flask to stop trypsinization. Collect the media containing the detached cells into a 50 ml conical tube and spin for 10 min at 300 x g at room temperature.
   3. Aspirate the supernatant without disturbing the pellet, and then re-suspend the cells in 2 ml of fresh complete media by gently pipetting the solution using a P1000 pipet.
   4. Determine the number of cells using a hemocytometer and then re-suspend cells in complete medium at the desired density (e.g. 10⁵ cells/ml). Plate the cells onto the ECM-coated coverslips (e.g. 500 μl into each well of the 24-well plate). Add additional fresh complete media to each well for 3-5 days of incubation at 37 °C, or until cells grow to the desired visual confluence.
3. Treatment with Psychosine.
   1. Reconstitute the psychosine to a stock concentration (108.3 mM) in dimethyl sulfoxide (DMSO). Dilute this stock in 100% EtOH to make a working stock that can then be added into each well of 24-well plate to achieve a final concentration of 10 μM.
   2. At the time of adding the psychosine, gently swirl the culture plate to mix the psychosine into culture media. Supplement the psychosine by adding the equivalent of 10 µM every other day for one week.
4. After 7 days of psychosine treatment, collect the media and pipet 500 µl of cold 4% paraformaldehyde (PFA) into each well. Incubate at room temperature for 20 min to fix the cells. Then, wash each well 3x with PBS and then keep plates at 4 °C until processed for immunocytochemistry (see below).
   NOTE: cell culture media can be collected for enzymatic or ELISA assay at this point if desired.

3. Immunocytochemistry (ICC) to Visualize Globoid Cells

1. Replace PBS with blocking buffer [PBS + 0.3% Tween-20 + 5% normal goat serum (NGS)], and incubate the cells for 1 hr at room temperature.
2. Incubate the fixed cells in primary antisera against Iba-1 [(1:1,000) diluted in PBS + 0.3% Tween-20 + 2% NGS] for at least 1 hr at room temperature, then wash each well with PBS 3x 5 min/wash.
3. Add the appropriate secondary fluorescence-conjugated antibody to identify the primary antibody labeling and counter-stain with 4',6-diamidino-2-phenylindole (DAPI, 1:1,000) to identify nuclei. Incubate in secondary antibody solution for 1 hr at room temperature and wash with PBS 3x 5 min.
4. Mount each coverglass to microscope slides using a mounting medium. View slides on a fluorescent microscope for visual and quantitative analyses.

4. Analysis and Characterization of Globoid Cells in Primary Culture

1. Use the following three morphological criteria to identify a globoid cell in culture:
   1. Identify globoid cells using fluorescent microscopy by immunopositive labeling for the microglial marker, Iba-1+.
   2. Identify globoid cells as polynucleated (i.e. containing two or more DAPI positive nuclei).
   3. Identify globoid cells by a rounded morphology distinct from the highly branched appearance of resting microglial cells.
      NOTE: Cells may also be collected for cell surface marker analysis and quantification by flow cytometry from these cultures.
2. Measuring the phagocytic activation of microglia and globoid cells
   1. To assess the phagocytic activity of psychosine-treated microglia, add FITC-labeled latex beads to the wells in accordance with the manufacturer’s instructions. Add fluorescent-conjugated latex beads 48 hr prior to fixation with PFA. Fix cells treated with beads (as mentioned in section 2.4), and process for immunocytochemistry (as described in section 3).
   2. Select fluorophore-conjugated secondary antibodies so that they do not overlap the fluorescent emission spectra of the fluorophore labeled beads.
   3. Analyze the phagocytotic profiles of Iba-1+ microglia by identifying those cells that co-localize with the fluorescently-labeled beads.
      NOTE: Psychosine will activate microglia. Phagocytosed beads will be identified within mononucleated and multinucleated Iba-1+ cells in this culture setting.

5. Induction of Globoid Cells from Purified Microglial Cultures

NOTE: An alternate approach to decipher intercellular signaling between the astrocyte and microglia is another advantage of this in vitro globoid cell model. Purification of primary microglial cells for replating or co-culturing can be achieved by their lower adherence property in culture, as described in the following steps (see Section 5.2).

1. Collect the media from the confluent mixed glial cultures into 50 ml conical tube and maintain it at 37 °C in the incubator.
2. Microglial Isolation from Primary Mixed Glial Cultures.
   1. After removing the media from a confluent mixed glial culture T175 flask, add warm shaking media [consisting of complete media + 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) + 25 mM sodium bicarbonate (Sigma)]. Shake the flasks for 3-4 hr in an orbital shaker at 100 rpm (37 °C). Collect the media from these shaken flasks into 50 ml conical tubes.
      NOTE: This media will include the less adherent microglia from the flasks.
   2. Spin the media at 300 x g for 10 min at room temperature. Aspirate the supernatant and then re-suspend the cells in 2 ml of the astrocyte-conditioned media (collected in step 5.1).
   3. Establish the number of cells acquired using a hemocytometer and then plate the microglial cells onto substrate-coated coverslips (see section 2.1) for immunocytochemistry, or plate onto UltraLow adherence well plates for future collection of globoid cells for co-culture with other cell types, such as oligodendrocytes (as described in the following section).
      NOTE: Once plated the microglia can be treated with psychosine (10 µM) as described above (see section 2.3).
3. Co-culture with Primary Oligodendrocytes for Analysis of Cytotoxicity.
   NOTE: Collection of psychosine-treated microglia from the low adherence plates for subsequent co-culture onto primary cultures of oligodendrocytes, or oligodendrocyte progenitor cells, can be used to examine the potential cytotoxic function of these globoid-like cells in vitro.
   1. Develop cultures of primary oligodendrocytes using established methods^18,20. To collect the psychosine-treated microglia, gently pipet to detach the loosely adherent cells from the bottom of the wells. Collect the cells, centrifuge at 300 x g for 10 min, and then carefully resuspend in oligodendrocyte differentiation media [composed of Neurobasal media, L-glutamine (1x), B-27 supplement (1x), and Triiodothyronine (T3, 10 ng/ml)].
   2. Count the number of cells using a hemocytometer and then seed the microglia onto the oligodendrocyte culture at 1 x 10⁵ cells/ml, resulting in an approximate 1:2 microglia/oligodendrocyte ratio. Incubate as a co-culture for up to 3 days.
   3. Fix the cells for immunocytochemistry (see section 2.4). Collect cell culture media for ELISA or chemical analyses as needed.

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Wyniki

This protocol, as written, is expected to take approximately 36 days to complete from start to finish (See Figure 1: Experimental Workflow Scheme). It has been our experience that the development of 'globoid-like' cells in this primary culture system is both reliable and reproducible: the formation of multinucleated cells in response to psychosine is consistently observed with 7 days of treatment.

Immunocytochemical staining of microglia using Iba-1 in conjunction with...

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Dyskusje

The protocol described herein provides a new model system in which to study the development and functional characterization of activated microglia and globoid cells. Prior work by Im et al. using a HEK293 cell line provided a template for the development of the present protocol for the study of globoid cell formation²¹. It is also important to point out that the globoid cells derived in the model differ from the native globoid cells identifiable in GLD. For instance, we have routinely observed quadran...

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Materiały

Name	Company	Catalog Number	Comments
Hank’s balanced salt solution (HBSS) containing no cations (Mg2+ and Ca2+).	Life technologies	14175-095
Neural Tissue Dissociation Kit	Miltenyi	130-092-628
40 μm cell-strainer	Fisherbrand	22363547
Hank’s balanced salt solution (HBSS) containing cations (Mg2+ and Ca2+).	Gibco	14025-092
Dulbecco's modified eagle medium (DMEM)	Gibco	11995-065
fetal bovine serum (FBS)	Atlanta Biologicals	S11150
Penicilin/Streptomycin	Life technologies	15070-063
Laminin	Sigma	L2020
Trypsin-EDTA solution	Life technologies	25299-056
Psychosine	Sigma	P9256
Dimethyl sulfoxide (DMSO)	Sigma	D2650
Paraformaldehyde (PFA)	Electron Microscopy Science	19208
Normal Goat Serum (NGS)	Invitrogen	PCN5000
Iba-1	WAKO	019-19741
Alexa Fluor conjugated antisera	Life Technologies	Various
Mounting Media	Southern Biotech	OB100-01
Phagocytic Assay Kit	Cayman Chemicals	500290
HEPES	Sigma	BP310-500