Intricate interplay between astrocytes and motor neurons in ALS

ALS results from the selective and progressive degeneration of motor neurons. Although the underlying disease mechanisms remain unknown, glial cells have been implicated in ALS disease progression. Here, we examine the effects of glial cell/motor neuron interactions on gene expression using the hSOD1^G93A (the G93A allele of the human superoxide dismutase gene) mouse model of ALS. We detect striking cell autonomous and nonautonomous changes in gene expression in cocultured motor neurons and glia, revealing that the two cell types profoundly affect each other. In addition, we found a remarkable concordance between the cell culture data and expression profiles of whole spinal cords and acutely isolated spinal cord cells during disease progression in the G93A mouse model, providing validation of the cell culture approach. Bioinformatics analyses identified changes in the expression of specific genes and signaling pathways that may contribute to motor neuron degeneration in ALS, among which are TGF-β signaling pathways.ALS is a late-onset, fatal neurodegenerative disease caused by the selective loss of upper and lower motor neurons in the brain and spinal cord and progressive paralysis of voluntary muscles; death ultimately results from respiratory failure (reviewed in ref. 1). Most ALS cases (∼90%) are sporadic, with an unknown cause, whereas the remaining cases are of familial origin (reviewed in ref. 2), among which ∼20–25% are caused by dominantly inherited mutations in the SOD1 gene; this gene encodes a cytosolic Cu/Zn superoxide dismutase (3). Overproducing pathogenic alleles of human SOD1 in mice and rats leads to late-onset progressive motor neuron degeneration, strikingly similar to the human disease (4–7). Because the pathological progression in both sporadic and familial ALS is indistinguishable (8, 9), insights derived from studies of the SOD1 mouse model are thought to be informative for both sporadic and familial ALS pathology.The fundamental pathological basis for ALS remains to be determined along with the specific insults that target motor neurons for death. Mutant SOD1 genes are expressed ubiquitously in humans and mice and when expressed exclusively in mouse motor neurons, are not sufficient to cause disease (10–12). An important insight into this enigma was provided by the observation that the presence of mutant SOD1 within neighboring nonneuronal cells contributes to motor neuron toxicity and thereby, disease onset and progression (reviewed in ref. 13). The principal nonneuronal cell types implicated in motor neuron death in ALS are astrocytes, microglia, and oligodendrocytes; in vivo approaches focused on excising the mutant transgene from microglia and astrocytes in SOD1-based ALS mouse models have shown that disease onset and/or progression are affected (reviewed in ref. 13). There is increasing evidence that the presence of the mutant SOD1 protein in these nonneuronal cell types contributes significantly to ALS disease progression in the ALS mouse model. Evidence that astrocytes also play a negative role in human ALS was provided by a recent study showing that astrocytes generated from postmortem spinal cords from SOD1 or sporadic ALS patients adversely affect the viability of cultured ES cell-derived mouse motor neurons (14).The question of extrinsic vs. intrinsic effects on gene expression in motor neurons in ALS in vivo has been difficult to address using laser capture microdissection (LCMD), because only the cell soma is captured, excluding the dendritic arbor as well as the axon. Moreover, LCMD is limited to neuronal cells, because it is not possible to cleanly capture glial cell bodies from among the neuropil in the spinal cord. Other approaches involve studies of entire spinal cords, which are heterogeneous and do not provide cell type-specific information. Thus, either approach alone yields an incomplete picture. Consequently, it has thus far not been possible to relate progressive gene expression changes in motor neurons to changes in gene expression in the surrounding glial cells in whole-animal studies. A potential solution to this problem is to make use of cell culture models to study how glial cells adversely affect motor neuron viability.In previous studies, we (15) and others (6) established a cell culture system to study astrocyte/motor neuron interactions. This approach involves the generation of motor neurons by in vitro differentiation of ES cells derived from mice harboring the human SOD1^G93A transgene. These ES cell-derived motor neurons are cocultured with either ES cell-derived or primary glial cells. Remarkably, motor neurons in this coculture system recapitulate aspects of the abnormal pathology characteristic of ALS in humans as well as in transgenic mice (15). In addition, SOD1^G93A mutant glia can adversely affect the viability of mutant as well as WT motor neurons in vitro and in vivo (14, 16).Although each approach to the analysis of ALS disease mechanisms (postmortem ALS patient samples, mouse models, and cell culture) has its limitations, an integrated approach that combines whole-animal and cell culture analyses could provide novel insights into the pathways leading to motor neuron-specific degeneration. Here, we describe an adaptation of this in vitro model to capture both cell autonomous and nonautonomous changes in neuronal and glial cell gene expression. We studied FACS-purified ES cell-derived motor neurons in sandwich cultures (17), in which motor neurons plated on glass coverslips are cultured over primary glial cells as a function of time. We then used separate RNA sequencing (RNA-Seq) analysis of the two cell types to identify changes in gene expression profiles intrinsic to each cell type (cell autonomous effects) or mediated by the cocultured cell (cell nonautonomous effects).In parallel, we used RNA-Seq to determine expression profiles of a longitudinal series of whole spinal cords from the same G93A mouse model of ALS over the course of the disease. These data, combined with gene expression data from acutely isolated glia, microglia, and oligodendrocytes from control and mutant SOD1 mouse spinal cords and ALS patient postmortem human spinal cord samples, may lead to the identification of common features that will provide an ALS disease signature and thereby, identify potential ALS drug targets.