Appearance of highly proliferative pathogenic astrocytes coincides with rapid paralysis in ALS mice.

Investigators have discovered what they claim is a previously unknown type of astrocyte in a mouse model of amyotrophic lateral sclerosis (ALS), which appears to drive the motoneuron death associated with the disease. These aberrant astrocytes, or AbA cells, isolated from the spinal cords of symptomatic ALS mice, express typical astrocyte markers, but demonstrate increased proliferation, lack of replicative senescence, and secrete soluble factors that induced motoneuron death with a 10-fold higher potency than the astrocytes isolated from neonatal ALS mice.

Reporting in PNAS, Luis Barbeito, Ph.D., at the Institut Pasteur de Montevideo in Uruguay, Joseph Beckman, Ph.D., at the Linus Pauling Institute at Oregon State University, and colleagues, suggest Aba cells could represent a therapeutic target for the future treatment of ALS. Their paper is titled “Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis.”

ALS is a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1), and is characterized by the loss of motoneurons and reactocytosis, the researchers explain. Mice and rats expressing human Cu-Zn superoxide dismutase (SOD1) mutations also develop a motor syndrome with symptoms and pathological features of the human disease, and rats expressing the SOD1G93A mutation, astrogliosis is associated with the disappearance of ventral motoneurons and a marked loss of the astrocytic glutamate transporter 1 (GLT1). Human astrocytes derived from the spinal cord of patients with sporadic and familial ALS also can kill motoneurons in culture.

However, studies in animal models have shown that selective ablation of either proliferating GFAP-expressing astrocytes or microglia (which also become activated in the spinal cords) fails to modify disease progression, and this suggests that there may be a different glial cell type at work that specifically contributes to motoneuron pathology. However, the researchers note, “it remains unknown whether all astrocytes are intrinsically neurotoxic for motoneurons or whether instead toxicity is restricted to a specific subclass of astrocytes.”

To look into this further by isolating glial cell populations present in ALS, they attempted to establish primary cultures from the spinal cords of both symptomatic SOD1G93A ALS rats (transgenic, or Tg animas), and their wild-type littermates (non-Tg rats). While the wild-type cultures yielded only a few cells that proliferated slowly and didn’t survive subsequent passages, cultures from the SOD1G93A rats yielded numerous cells that proliferated rapidly in vitro and formed clusters of elongated flat cells resembling astrocytes. Putting an agarose layer on top of these cells further stimulated the further proliferating potential of Tg astrocytes, but not the non-Tg astrocytes, and after six days, the astrocytic phenotype in the Tg cultures markedly increased and organized into a monolayer bearing sparse microglial cells.

Tg-derived cultures grown in defined medium to oligodendrocytes yielded by NG2 cells and also S100β+ astrocytes, which demonstrated that “AbA cells do not arise from the NG2 oligodendrocyte precursors also known to proliferate in the spinal cord of symptomatic ALS mice,” the authors remark.

They then moved on to evaluate the expression of astrocytic markers in AbA cell cultures from subsequent passages, by comparison wtih primary spinal cord astrocytes from neonatal SODIG93A rats and non-Tg littermates. While both AbA cells and neonatal astrocytes expressed most of the typical astrocytic markers, such as GFAP, vimentin, S100β, connexion 43 (Cx43; a gap junction protein), and glutamine synthase, the Tg rat-derived AbA cells didn’t appear to produce GLT1 protein, and NG2 was only expressed after passage 4. AbA cells also displayed only weak and diffuse perinuclear GFAP labeling, but intense staining for S100β, while neonatal astrocytes exhibited filamentous GFAP but only low S100β expression. Interestingly, AbA cells grew much faster through passage 10 than Tg and non-Tg neonatal astrocytes, and even continued increasing after reaching confluence, which suggested there was a defect in contact inhibition.

The toxicity of AbA cells to motoneurons was demonstrated by plating embryonic motoneurons on top of confluent AbA cells. While just 10% of motoneurons survived co-culturing with AbA cells for two days, 60% of neurons co-cultured with Tg cells survived for two days, and 100% of neurons co-cultured wth non-Tg neonatal astrocytes survived. The increased cell death mediated by Aba cells appeared to be caused by soluble secreted factors, as adding conditioned medium (CM) from the Aba cell cultures to embryonic motoneuron cultures maintained with glial cell-derived neurotrophic factor (GDNF) lead to at least a 10-fold higher motoneuron death rate than CM taken from neonatal Tg astrocytes. CM from neonatal non-Tg astrocytes had no effect on the survival of cultured embryonic motoneurons. “AbA cells display an unprecedented toxicity to motoneurons that greatly exceeds that of neonatal Tg astrocytes expressing mutant SOD1,” they add. “Notably, the neurotoxicity of AbA CM was specific to motoneurons, because even a 1:10 dilution failed to kill primary cultures of embryonic hippocampal neurons.”

The team subsequently used immunohistochemical staining to look for AbA-like cells in the degenerating spinal cords of SOD1G93A rats, and in the spinal cords of non-Tg and asymptomatic Tg animals. While S100β staining was either low or moderate in the non-Tg and asymptomatic rats, it was dramatically increased in symptomatic Tg rats, and localized in the nuclei and cytoplasm of a hypertrophic astrocytes that expressed GFAP just in cell bodies and proximal processes. Importantly, these cells were most often located in the spinal cord near damaged motoneurons, and at the interface between gray and white matter. Cx43 staining also increased markedly in symptomatic rats, and co-localized with S100β hypertrophic astrocytes. “Such astrocytes appeared at the time of disease onset, and their number increased sharply at disease end stage,” the researchers report.

Of particular interest was the finding that S100β didn’t co-localize with NG2 oligodendrocyte precursors that have previously been described in ALS mice, suggesting that the S100β+ cells constitute a different cell population.

The overall results suggest not only that Aba cells are a distinct subpopulation of highly toxic astrocytes that aren’t derived from NG2 oligodendrocyte progenitors, but also indicate “a link between the appearance of pathogenic AbA cells and the rapid progression of paralysis characteristic of the SOD1G93A rat model,” the authors conclude. “Cultured AbA cells are almost undistinguishable morphologically from primary neonatal astrocytes and exhibit a set of distinctive antigenic markers of undifferentiated astrocytes including high S100β and Cx43 expression and low levels of nonfilamentous GFAP…Although the mechanism of AbA neurotoxicity is under active investigation, the possibility exists that AbA cells produce cytokines, excitotoxins, or trophic factors such as nerve-growth factor that may kill motoneurons specifically.” 

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