Poplawski, G. H. D. | bioRxiv Science

bioRxiv2026

Clinical Relevant Immunosuppressive Drugs Differentially Modulate Axonal Outgrowth from Human Stem Cell Derived Neurons

Neural stem cell (NSC) transplantation is a promising strategy for repairing the injured spinal cord, but transplanted cells typically require immunosuppressive therapy to prevent rejection--even for induced pluripotent stem cell (iPSC)-derived autologous grafts. However, the effects of immunosuppressive drugs on neurite outgrowth and axonal regeneration-- processes critical for neural circuit reconstruction--have not been fully characterized. In this study, we tested nine clinically relevant immunosuppressants on human iPSC-derived neurons and primary human spinal cord NSCs in vitro at concentrations approximating clinical exposure levels. The drug panel included FK-506 (tacrolimus), cyclosporine A (CsA), rapamycin, belatacept (Nulojix), etanercept (Enbrel), mycophenolate mofetil (CellCept), cyclophosphamide (Cytoxan), prednisone, and azathioprine (Imuran). Neurite outgrowth was quantified via automated high-content imaging. Multiple agents, including CsA, Imuran, Nulojix, and CellCept, induced significant reductions in neurite outgrowth in a cell type- and dose-dependent manner, with CsA producing the most robust and consistent inhibition across both cell lines. In contrast, FK-506 showed no significant effect on neurite extension at clinically relevant concentrations. Consistent with the in vitro results, human neural progenitor cell grafts in a rodent spinal cord injury model exhibited significantly reduced graft-derived axon extension in the host spinal cord when hosts were treated with CsA rather than FK-506. These findings demonstrate that immunosuppressant choice can profoundly influence neural graft integration and axonal regeneration. Our study underscores the importance of preclinical evaluation of immunosuppressive regimens and suggests that selecting agents such as FK-506 over CsA may improve outcomes in future stem cell-based therapeutic trials for spinal cord injury and related disorders of the central nervous system. HighlightsO_LICyclosporine A inhibits axon outgrowth in human neurons in vitro C_LIO_LIFK-506 preserves neurite extension across stem cell-derived neuron types C_LIO_LIAxon outgrowth is reduced in vivo with CsA but not FK-506 C_LIO_LIImmunosuppressant selection critically affects neural graft integration C_LIO_LIFK-506 may be preferable to CsA for SCI cell transplantation protocols C_LI

neuroscience↗

bioRxiv2025

Cell cycle dysregulation contributes to neurodegeneration in humanneurons from ALS/FTD-related C9orf72 repeat expansion carriers.

The C9orf72 hexanucleotide repeat expansion GGGGCC (G4C2) cause the most common genetic forms of ALS and frontotemporal dementia, affecting thousands of patients worldwide with uniformly fatal outcomes. C9orf72 ALS/FTD patients lack targeted treatments because druggable molecular vulnerabilities remain unidentified. Using iPSC-derived motor neurons from C9orf72 carriers and age-matched controls, we performed comprehensive cell cycle analysis, drug screening, and single-nucleus RNA sequencing validation in human brain tissue. C9orf72 neurons exhibit age-dependent cell cycle reentry with increased S-phase cells, elevated cyclin and CDK expression, and aberrant cell cycle gene signatures confirmed in patient brain excitatory neurons. Mechanistically, arginine-containing dipeptide repeat proteins (poly-GR, poly-PR) drive this cell cycle activation through CDK4/6 pathway stimulation, while C9orf72 loss-of-function alone shows no effect. Critically, the FDA-approved CDK4/6 inhibitor palbociclib normalizes cell cycle progression, reduces S-phase entry, and rescues neuronal survival with significant reduction in motor neuron death. Single-nucleus RNA-sequencing analyses from C9orf72 patient cortex reveals cell cycle-activated neuronal subclusters. Copy number variation, gene ontology and pathways analyses revealed alterations in DNA repair pathways, cell cycle regulation and cell cycle transition, validating our in vitro findings. These results identify cell cycle dysregulation as a therapeutic target in C9orf72 ALS/FTD with clinical translation potential using existing therapeutics.

neuroscience↗