ALS mutations do not alter perineuronal net formation in human stem cell-derived motor neurons

Perineuronal nets (PNNs) are extracellular matrix structures that stabilise synaptic inputs and play a role in regulating neuronal plasticity. Although PNN dysregulation is observed in several neurological disorders, their relevance to amyotrophic lateral sclerosis (ALS) remains unclear. In particular, the extent to which PNN alterations reported in ALS animal models are motor neuron (MN)-intrinsic is unknown. We investigated whether human pluripotent stem cell-derived MNs form PNN-like structures in vitro, and whether ALS-associated mutations alter this process. We show that PNN-like structures containing hyaluronan, tenascin-R, and aggrecan form in in vitro co-cultures of iPSC-derived MNs and astrocytes, and that their formation and gene expression were not altered by ALS mutations. To explore whether PNN dysregulation reflects contributions from other cell types or selective MN vulnerability, we conducted meta-analyses of transcriptomic datasets from pluripotent stem cell-derived astrocytes carrying ALS-associated mutations, as well as datasets comparing MN populations with differential susceptibility to ALS. These analyses revealed no consistent differences in PNN-related gene expression within human stem cell-derived MNs. In contrast, transcriptomic analyses of human post-mortem ALS tissues revealed dysregulation of PNN-related genes, including core PNN components. Taken together, these findings indicate that PNN-related alterations described in ALS animal models are not reproduced by ALS-associated mutations in MNs alone, and instead point to a role for additional cellular components within the central nervous system.