Viral mediated knockdown of GATA6 in SMA iPSC-derived astrocytes prevents motor neuron loss and microglial activation

Abstract

Spinal muscular atrophy (SMA), a pediatric genetic disorder, is characterized by the profound loss of spinal cord motor neurons and subsequent muscle atrophy and death. Although the mechanisms underlying motor neuron loss are not entirely clear, data from our work and others support the idea that glial cells contribute to disease pathology. GATA6, a transcription factor that we have previously shown to be upregulated in SMA astrocytes, is negatively regulated by SMN and can increase the expression of the inflammatory regulator NFκB. In this study, we identified upregulated GATA6 as a contributor to increased activation, pro-inflammatory ligand production, and neurotoxicity in spinal-cord patterned astrocytes differentiated from SMA patient induced pluripotent stem cells. Reducing GATA6 expression in SMA astrocytes via lentiviral infection ameliorated these effects to healthy control levels. Additionally, we found that SMA astrocytes contribute to SMA microglial phagocytosis, which was again decreased by lentiviral-mediated knockdown of GATA6. Together these data identify a role of GATA6 in SMA astrocyte pathology and further highlight glia as important targets of therapeutic intervention in SMA.

Diminished motor neuron activity driven by abnormal astrocytic GLAST glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery

“…Overall, our work defines a novel SMN-associated disease mechanism involving abnormal glutamate transporter activity and regulation in astrocytes that drives motor neuron inactivity in SMA. In vivo , astrocytic-driven dysfunction is likely to occur in combination with intrinsic motor neuron defects and sensory neuron-mediated dysfunction, with potential input from other glial cell types including microglia (15, 50, 88). Studies have already begun to test the efficacy of therapeutic compounds in order to increase motor neuron activity at the central afferent synapse (89, 90).…”

“…We next wanted to determine if human spinal cord-like astrocytes, a more disease relevant cell population for studying SMA pathology, similarly display intrinsic defects in synaptic neuromodulation which could directly impact motor neuron activity. Ventral-caudal patterned motor neurons (53) and astrocytes (50) were derived from healthy and patient iPSCs before seeding together onto microelectrode array (MEA) plates (Figure 2A). The differentiation protocols used in this study allowed for the initial, independent generation of motor neurons and astrocytes from iPSC lines.…”

“…iPSC maintenance and differentiation iPSC lines were cultured on Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix or Geltrex ™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Gibco) coated 6-well plates in Essential 8 medium (Gibco) and were passaged every 3 days using Versene (EDTA-based, Gibco) when 80% confluent. iPSCs were differentiated into spinal cord-like motor neurons or astrocytes using protocols recently described in (50). Neural progenitor cells (NPCs) in the form of embryoid body aggregates (for motor neuron differentiation) or monolayer cultures (for astrocyte differentiation) were generated from iPSCs through dual SMAD inhibition (SB 431542 and LDN 1931899 (biogems) and Wnt activation (Chir 99021 (biogems).…”

Microglia Influence Neurofilament Deposition in ALS iPSC-Derived Motor Neurons

“…Spinal cord patterned astrocytes were generated from iPSC-derived neural progenitor cells (NPCs) [ 38 ]. Differentiation reagents were purchased from ThermoFisher unless otherwise noted.…”

Microglia influence neurofilament deposition in ALS iPSC-derived motor neurons

“…Spinal cord patterned astrocytes were generated from iPSC-derived neural progenitor cells (NPCs) [38]. Briefly, iPSCs were grown to confluency, dissociated with Accutase, and plated at 2 million cells/well into Matrigel (Corning) coated 6 well-plates in NPC base medium (50% DMEM/ F12, 50% Neurobasal, 2% B27, 1% N2, 1% Antibiotic/ Antimycotic, 0.1% B-mercaptoethanol, 50ng/mL Laminin, and 0.5uM Ascorbic Acid) supplemented with 10uM Y-27632, 3uM Chir-99021, 40uM SB431542, and 0.2uM LDN193189.…”