Spinal astrocyte dysfunction drives motor neuron loss in late-onset spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder caused by a loss of the survival of motor neuron 1 (SMN1) gene, resulting in a loss of spinal motor neurons (MNs), leading to muscle weakness and wasting. The pathogenesis of MN loss in SMA and the selective vulnerability in different cellular populations are not fully understood. To investigate the role of spinal astrocytes in the pathogenesis of late-onset SMA, we used a mouse model in addition to in vitro approaches. Immunostaining, Western blot analysis, small interfering ribonucleic acid (siRNA) transfections, functional assays, enzyme-linked immunosorbent assay (ELISA), behavioral tests, and electrophysiological measurements were performed. Early activation of spinal astrocytes and a reduction of the excitatory amino acid transporter 1 (EAAT1) on postnatal day (P) 20 preceded the loss of spinal MNs in SMA mice occurring on P42. EAAT1 reduction resulted in elevated glutamate levels in the spinal cord of SMA mice at P20 and P42. SMA-like astrocytes generated by siRNA and an ex vivo model of glutamate excitotoxicity involving organotypic spinal cord slice cultures revealed the critical role of glutamate homeostasis in the degeneration of MNs. The pre-emptive administration of arundic acid (AA), as an inhibitor of astrocyte activation, to SMA mice prior to the loss of motor neurons (P28) resulted in elevated EAAT1 protein levels compared to vehicle-treated SMA mice and prevented the increase of glutamate in the spinal cord and the loss of spinal MNs. Furthermore, AA preserved motor functions during behavioral experiments, the electrophysiological properties, and muscle alteration of SMA mice. In a translational approach, we transfected healthy human fibroblasts with SMN1 siRNA, resulting in reduced EAAT1 expression and reduced uptake but increased glutamate release. These findings were verified by detecting elevated glutamate levels and reduced levels of EAAT1 in cerebrospinal fluid of untreated SMA type 2 and 3 patients. In addition, glutamate was elevated in serum samples, while EAAT1 was not detectable. Our data give evidence for the crucial role of spinal astrocytes in the pathogenesis of late-onset SMA, a potential driving force for MN loss by glutamate excitotoxicity caused by EAAT1 reduction as an early pathophysiological event. Furthermore, our study introduces EAAT1 as a potential therapeutic target for additional SMN-independent therapy strategies to complement SMN-enhancing drugs.

Modulation of Glutamate Transporter EAAT1 and Inward-Rectifier Potassium Channel Kir4.1 Expression in Cultured Spinal Cord Astrocytes by Platinum-Based Chemotherapeutics.

Platinum-based chemotherapeutics still play an essential role in cancer treatment. Despite their high effectiveness, severe side effects such as chemotherapy-induced neuropathy (CIPN) occur frequently. The pathophysiology of CIPN by platinum-based chemotherapeutics is not fully understood yet, but primarily the disturbance of dorsal root ganglion cells is discussed. However, there is increasing evidence of central nervous system involvement with activation of spinal cord astrocytes after treatment with chemotherapeutics. We investigated the influence of cis- or oxaliplatin on the functionality of cultured rat spinal cord astrocytes by using immunocytochemistry and patch-clamp electrophysiology. Cis- or oxaliplatin activated spinal astrocytes and led to downregulation of the excitatory amino acid transporter 1 (EAAT1) expression. Furthermore, the expression and function of potassium channel Kir4.1 were modulated. Pre-exposure to a specific Kir4.1 blocker in control astrocytes led to a reduced immune reactivity (IR) of EAAT1 and a nearly complete block of the current density. When spinal astrocytes were pre-exposed to antibiotic minocycline, all effects of cis- or oxaliplatin were abolished. Taken together, the modulation of Kir4.1 and EAAT1 proteins in astrocytes could be linked to the direct impact of cis- or oxaliplatin, identifying spinal astrocytes as a potential target in the prevention and treatment of chemotherapy-induced neuropathy.

Induction of Survival of Motor Neuron (SMN) Protein Deficiency in Spinal Astrocytes by Small Interfering RNA as an In Vitro Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a motor neuron disorder leading to progressive loss of ventral horn neurons resulting in muscle wasting. Here we investigate the contribution of spinal astrocytes to the pathogenesis of late-onset SMA forms using a mouse model. Furthermore, we generated SMA-like astrocytes using survival of motor neuron (SMN) siRNA transfection techniques. In the SMA mouse model, the activation of spinal astrocytes and the reduction of the inward rectifier potassium channel Kir4.1 and excitatory amino acid transporter 1 (EAAT1) were observed at postnatal day (P) 28, preceding the loss of spinal motor neurons appearing earliest at P42. Using SMA-like astrocytes, we could mimic the modulation of spinal astrocytes of the mouse model in a dish and perform electrophysiological assessments and functional assays. In SMA-like astrocytes, glutamate uptake was diminished due to a reduction in EAAT1. Furthermore, patch-clamp measurements revealed reduced potassium uptake into astrocytes with membrane depolarization. Additionally, exposure of healthy spinal motor neurons to a conditioned medium of SMA-like astrocytes resulted in increased firing frequency. These data demonstrate spinal astrocytes' crucial role in the late-onset SMA forms' pathogenesis.