Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis

Abstract

Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as "AbA cells") from primary spinal cord cultures of symptomatic rats expressing the SOD1(G93A) mutation. Isolation was based on AbA cells' marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1(G93A) astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.

Fig. 1.

Establishment of AbA cell cultures. Representative phase-contrast microphotographs of the establishment of primary spinal cord cultures prepared from symptomatic SOD1^G93A rats or non-Tg littermates (Insets). The cells were plated in a culture flask and observed under a phase-contrast microscope at 2, 7, and 16 days in vitro (DIV) and after replating at passage 12. Note the increased cell number in the culture of the symptomatic rat spinal cord compared with the culture from the non-Tg littermate. Microglial cells (arrows) disappeared progressively with successive passages. (Scale bars: 50 μm.)

Fig. 2.

Expression of astrocyte markers and proliferation in AbA cells. Cultures of AbA cells were compared with non-Tg or SOD1^G93A Tg primary neonatal spinal cord astrocytes. (A) Confocal imaging of cells immunostained against GFAP (green) and S100β (red) (Upper) and Cx43 (green) (Lower). In upper panels, yellow staining indicates cells expressing both GFAP and S100β. (Scale bars: 50 μm.) Note AbA cells’ low GFAP immunoreactivity but increased S100β and Cx43 immunoreactivity compared with neonatal astrocytes. (B) Western blotting analysis of astrocytic markers in AbA cells compared with primary astrocytes. AbA cells’ differential protein expression pattern is evidenced by decreased GFAP, absent GLT1, and augmented Cx43. (C) Growth of AbA cells (passage 10) and primary neonatal astrocytes was assessed over 9 d. The growth rate of AbA cells was almost twice that of primary astrocytes.

Fig. 3.

AbA cells specifically induced motoneuron death. (A) Embryonic motoneurons were seeded on top of confluent feeder layers of neonatal astrocytes or AbA cells, and survival was assessed 48 h later. Note that the survival rate of motoneurons maintained on top of AbA cells was <10% but was 100% for non-Tg (100%, dotted line) and 60% for Tg neonatal astrocytes. (B) CM from neonatal astrocytes or AbA cells was added to pure motoneurons 24 h after plating. The final fold dilution is indicated in each condition. CM from non-Tg astrocytes did not induce motoneuron death; thus it was taken as a control (100%, dotted line). Note that CM from AbA cells exerted significant motoneuron loss at dilutions up to 1:1,000, whereas Tg astrocyte CM was neurotoxic at dilutions up to 1:100. (C) Lack of neurotoxic activity of increasing dilutions of AbA CM in primary cultures of hippocampal neurons. Data are shown as mean ± SD; in A and B, ^#P < 0.01 and *P < 0.05 with respect to CM from non-Tg astrocytes.

Fig. 4.

Identification of AbA-like cells in the degenerating spinal cord. (A) Representative microphotographs of GFAP (red), S100β (green), and Cx43 (red) immunostaining in lumbar spinal cord sections from non-Tg, asymptomatic (Tg-Asymp), and symptomatic (Tg-symp) SOD1^G93A rats. Dotted lines in the top row indicate the border between gray and white matter in low-magnification representative microphotographs. The perimeter of large motoneurons has been drawn in the middle and bottom rows. Note that S100β was up-regulated in the spinal cord of symptomatic rats and especially was expressed in a population of hypertrophic cells with astrocyte morphology. Most of these cells displayed colocalization of S100β and GFAP (yellow). Cx43 immunoreactivity also was increased in Tg-symp spinal cords, being colocalized with S100β in most hypertrophic AbA-like cells (yellow in bottom row). (Scale bars: 50 μm in GFAP/S100β and 20 μm in Cx43/S100β.) (B) Increased number of AbA-like cells in the ventral horn of Tg rats during the progression of the disease. Inset shows the ventral horn area analyzed. Data are shown as mean ± SD; *P < 0.05. (C) Representative confocal immunostaining against NG2 and S100β in the ventral horn of a Tg-symp rat showing that AbA cells are not stained for NG2. (Scale bar: 20 μm.)