Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line.

Abstract

Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) MATERIALS AND METHODSCell Culture. Parental embryonic day 15 rat ventral spinal neuron preparation, mouse N18TG2 neuroblastoma culture, somatic cell fusion, and hybrid selection were performed as described (25,26). VSC 4.1 hybrid cells were maintained in logarithmic-phase growth on poly(L-ornithine)-precoated T-75 flasks (Coming) in Dulbecco's modified Eagle's medium/F-12 growth medium (GIBCO) containing Sato's components (Sigma) and 2% heat inactivated newborn calf serum (HyClone). VSC 4.1 cells undergoing cAMP-induced differentiation were plated at 1.2-1.5 x 106 cells per 75-mm2 flask and treated for 7 days with 1 mM dibutyryl cAMP or 1 mM 8-bromo-cAMP before use. Aphidicolin (0.4 pg/ml) (27) (Sigma) was added for 2 days after 24 hr in cAMP, and after 2 additional days of aphidicolin-free growth surviving cells were maintained in medium containing 1 mM cAMP and 0.025-0.05 ,ug of aphidicolin per ml. Differentiated hybrids were harvested by trituration after 1-hr incubation at 370C in Ca2+/Mg2+-free Hanks' balanced salt solution containing 1 mM EDTA and transferred into 24-well plates (Coming) at 2.0 x 104 cells per well [for direct cell count and lactate dehydrogenase (LDH) assays] or per 11-mm-diameter glass coverslip (for fluorescence assays). Pharmacologic agents and immunoglobulins were added to cells 24 hr after replating and were maintained in growth medium for the entire experAbbreviations: ALS, amyotrophic lateral sclerosis; LEMS, Lam-bert-Eaton myasthenic syndrome; VGCC, voltage-gated calcium channel; FDA, fluorescein diacetate; PI, propidium iodide; LDH, lactate dehydrogenase; AMPA, DL-a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.tPresent address:

Differential Localization of Voltage-Dependent Calcium Channel α1Subunits at the Human and Rat Neuromuscular Junction

“…In this disease it has been reported that a large proportion of patients has circulating autoantibodies directed against the ␣ 1 subunit of VDCCs Kimura et al, 1994). It is thought that VDCCs in MN terminals at the NMJ may represent targets for these autoantibodies and may play a role in MN degeneration in ALS Smith et al, 1994;Mosier et al, 1995;Siklos et al, 1996).…”

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

“…Differentiated I-VSC 4.1 cells expressing high levels of calbindin-D28K were significantly resistant to toxicity of ALS IgG (Fig. 6), which had been previously demonstrated to induce cell death of differentiated VSC 4.1 cells (7,15). After 48 h, the mean survival of differentiated I-VSC 4.1 cells incubated with ALS IgG was 98.1 + 13.1% compared to PBS-treated cultures.…”

“…In control experiments, the level of heavy-chain neurofilament protein mRNA as well as parvalbumin mRNA remained unchanged in I-VSC 4.1 cells. Similarly, the increase in neurofilament expression with cAMP differentiation reported in VSC 4.1 cells (15,24) (Fig. 4) godeoxynucleotide (E).…”

“…Statistical analysis of the ALS IgG-mediated toxicity by using one-way ANOVA shows a significance of P < 0. (calbindin-D28K or parvalbumin) (7) through a mechanism that is calcium-dependent and possibly mediated through voltagegated calcium channels (15). ALS IgG can also increase calcium currents in motoneuron hybrid cells (28) and induces a transient increase in intracellular calcium (24) that correlates with subsequent apoptotic cell death (16).…”

“…Cell survival was assessed after incubation for 48 h with ALS IgG by counting five contiguous 1-mm2 fields predetermined and marked after plating at day zero. This direct counting assay has been well correlated with vital staining using fluorescein diacetate and propidium iodide and with the lactate dehydrogenase release assay (15).…”