Preservation of miR-9-5p and miR-124-3p in ALS-resistant oculomotor neurons contrasts with their downregulation in vulnerable spinal motor neurons, irrespective of TDP-43 pathology.

Selective vulnerability of motor neurons is a defining feature of amyotrophic lateral sclerosis (ALS) and provides a valuable framework for uncovering mechanisms that distinguish resilient from vulnerable neuronal populations. We investigated whether dysregulation of neuroprotective microRNAs (miRNAs), miR-9-5p and miR-124-3p, contributes to the differential susceptibility of motor neuron subtypes. We focused on cervical spinal motor neurons (SMNs), which undergo drastic degeneration in ALS, and oculomotor neurons (OMNs), which remain functionally intact and rarely degenerate, allowing preservation of eye movement in ALS patients. Using a modified multiplexed fluorescent in situ hybridization protocol combined with immunofluorescence, we quantified the expression of miR-9-5p and miR-124-3p in cervical SMNs and OMNs from ALS and control cases. We observed significant downregulation of both miRNAs in ALS SMNs, while their expression was maintained in ALS OMNs. Stratification of ALS SMNs by TDP-43 pathological status revealed similarly reduced miRNA expression in neurons with and without cytoplasmic inclusions, suggesting that miRNA downregulation occurs independently of visible TDP-43 pathology. We assessed the localization of the Dicer cofactor TRBP and found that it colocalized with TDP-43 inclusions in ALS SMNs, suggesting that TRBP sequestration could prevent proper miRNA processing. However, TRBP remained normally localized in neurons without cytoplasmic inclusions, indicating that sequestration cannot fully account for miRNA reduction across all ALS motor neurons. These findings support a model in which early or subtle disruptions, preceding visible pathology, may also contribute to miRNA downregulation in ALS. By identifying preserved miRNA networks as correlates of oculomotor neuron resilience in ALS, this work also exposes new therapeutic targets potentially capable of reinstating miRNA expression and reprogramming vulnerable SMNs.