INVESTIGATING TRIPARTITE SYNAPSE PATHOLOGY IN ALS UTILIZING A HIPSC-DERIVED ORGANOID MODEL

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by motor neuron (MN) degeneration. Early synaptic alterations and glial dysfunction are thought to contribute to MN degeneration. Previous research indicates the selective vulnerability of tripartite synapses in spinal cords from ALS mouse models and human post-mortem tissue. This leads us to hypothesise that tripartite synapse pathology represents a conserved hallmark of early-stage ALS.
To investigate this, we first used human induced pluripotent stem cell (iPSC)–derived cortical organoids. Using immunohistochemistry, we validated synaptic and astrocytic labelling in these organoids. Analysis of cortical organoids derived from human iPSCs harbouring C9ORF72 mutations (C9) and gene-corrected controls (C9Δ) showed no change in the proportion of synapses contacted by the astrocytic marker ezrin, indicating no selective loss of tripartite synapses. However, we observed a significant reduction in postsynaptic size between C9 and C9Δ lines, independent of whether synapses were tripartite or non-tripartite. Contrary to our hypothesis, these findings suggest that while synaptic structure is altered in ALS neurons, there is no preferential effect on tripartite synapses in cortical organoids.
These findings highlight the need to investigate whether such pathology occurs in the spinal cord. We have therefore generated and maintained spinal cord organoids up to 50 days in vitro and demonstrate their expression of MN markers (CHAT), and synaptic markers (PSD-95 and Synapsin). Our ongoing research using spinal cord organoids will help address the hypothesis of whether tripartite synapse pathology is a conserved hallmark of ALS that can be modelled with human iPSC technology.