Bunner, W. | 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

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bioRxiv2025

Lack of ADAP1/Centaurin-α1 Ameliorates Cognitive Impairment and Neuropathological Hallmarks in a Mouse Model of Alzheimer's Disease

ArfGAP, with dual PH domain-containing protein 1/Centaurin-1 (ADAP1CentA1), is a brain-enriched and highly conserved Arf6 GTPase-activating and Ras-anchoring protein. ADAP1 is involved in dendritic outgrowth and arborization, synaptogenesis, and axonal polarization by regulating the dynamics of the actin cytoskeleton. An increased level of ADAP1 and its association with amyloid plaques in the human Alzheimers disease (AD) brain suggest a role for this protein in AD progression. To understand the role of ADAP1/CentA1 in neurodegeneration, we crossbred CentA1 KO mice with the hAPP-J20 mouse model of AD (J20 x CentA1 KO). We then evaluated the gene expression profile and the behavioral and neuropathological hallmarks of AD to determine the impact of eliminating ADAP1/CentA1 expression on AD-related phenotypes. Spatial memory assessed by the Morris Water Maze test showed significant impairment in J20 mice, which was rescued by the deletion of CentA1. Neuropathological hallmarks of AD, such as deposits of amyloid plaques and neuroinflammation, were significantly reduced in the AD model mice with CentA1 KO background. To identify potential mediators of AD phenotype rescue, we analyzed differentially expressed genes (DEGs) between genotypes, by employing transcriptome profiling with Nanostring nCounter Neuropathology and Neuroinflammation panels. We found significant upregulation of genes associated with apoptosis and gliosis in the brain of J20 mice. However, many of these genes, including the pro-apoptotic gene, Bid, were restored in the brains of J20 x CentA1 KO mice compared to J20 mice. In summary, our data indicate that CentA1 is required for the progression of AD phenotypes and that targeting CentA1 signaling at mitochondria might have therapeutic potential for AD prevention or treatment. SIGNIFICANCE STATEMENTADAP1/Centaurin-1 (CentA1) is highly enriched in the brain and increased CentA1 level has been linked to Alzheimers disease (AD). However, the precise role of ADAP1 in the pathogenesis of AD is poorly understood. We found that genetic deletion of CentA1 in the AD model mice rescues the pathological hallmarks of AD, including loss of dendritic spines in the hippocampus, amyloid plaque deposition, neuroinflammation and spatial memory deficits. Transcriptome analysis of forebrain samples using NanoString nCounter panels for 880 genes, identified the pro-apoptotic protein, Bid, with significantly reduced expression in the J20 mice on ADAP1 KO background. These findings point towards a role of ADAP1 in the activation of the mitochondrial pathways of death associated with the progression of neurodegeneration.

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