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 (survival motor neuron) and can increase the expression of inflammatory regulator NFκB. In this study,

Diminished motor neuron activity driven by abnormal astrocytic EAAT1 glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery

“…Using a microelectrode array (MEA) approach, we first wanted to determine if SMA patient astrocytes possess intrinsic defects in synaptic neuromodulation that could drive motor neuron dysfunction. The initial, independent derivation of iPSC‐derived spinal cord‐patterned motor neurons (Maury et al, 2015) and astrocytes (Allison et al, 2022) allowed for the assessment of different healthy and SMA patient co‐culture combinations to determine if changes in neural activity are motor neuron‐intrinsic or driven by diseased astrocytes. Specifically, we assessed if motor neuron activity phenotypes could be attributed to astrocytic cellular interactions (direct contact motor neuron‐astrocyte co‐cultures) and/or secreted factors (astrocyte conditioned media [ACM] treated motor neurons).…”

“…Overall, our work defines a novel SMN‐associated disease mechanism involving abnormal astrocyte glutamate transporter activity that diminishes motor neuron activity in SMA. In vivo, astrocytic‐driven dysfunction is likely to occur in combination with intrinsic motor neuron defects and sensory neuron‐mediated dysfunction, with further perturbation likely happening in other glial cell types including microglia (Allison et al, 2022; Khayrullina et al, 2022; Tarabal et al, 2014). Studies have already begun to test the efficacy of therapeutic compounds in order to increase motor neuron activity at the central afferent synapse (Kovalchuk et al, 2018; Simon et al, 2021).…”

“…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 (Allison et al, 2022). 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]).…”

Systemic LPS Administration Stimulates the Activation of Non-Neuronal Cells in an Experimental Model of Spinal Muscular Atrophy

“…LPS systemic administration is known to rapidly promote pro-inflammatory microglia phenotypes in the CNS [91], and the bidirectional communication of these populations is a crucial mediator in terms of delaying or accelerating the establishment of neuroinflammation [92]. In SMA, aberrant communication of these populations may contribute to disease pathology [93]. In our study, LPS systemic administration was strongly correlated with an increase in CNS pro-inflammatory microglia and astrocytes (defined as Iba-1 + iNOS + and GFAP + C3 + cells, respectively), and this effect, although present in both SMN∆7 and control mice, was more prominent for mice with experimental SMA compared to control mice.…”

Survival motor neuron protein and neurite degeneration are regulated by Gemin3 in spinal muscular atrophy motoneurons

“…In the nervous system, neuronal activation of NF-κB is critical for cell survival and differentiation, whereas non-neuronal cell activation of NF-κB has a detrimental effect on neurons, triggering inflammatory and cell death mechanisms ( Mincheva-Tasheva and Soler, 2013 ). For instance, increased phosphorylation and upregulation of NF-κB was described in SMN-depleted macrophages ( Ando et al, 2020 ) and human iPSC-derived SMA astrocytes ( Allison et al, 2022 ), suggesting that SMN protein may regulate the activation of these cells and the inflammatory response in SMA. In cultured MNs, pharmacological inhibition of NF-κB pathway or endogenous RelA knockdown reduces SMN level ( Arumugam et al, 2018 ), suggesting an active role of NF-κB in SMN expression in these cells.…”