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

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 postnata....

Results

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 a nucle.......

Discussion

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.......

Materials

List of materials used in this article

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