Role of FK506 Binding Proteins in the Neuroregenerative Mechanism of Action of Locally Administered FK506 to the Peripheral Nerve
Kasra Tajdaran, MASc, PhD, Jennifer J Zhang, MD, PhD, Gregory H. Borschel, MD, FAAP, FACS and Tessa Gordon, PhD, Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, Toronto, ON, Canada
Purpose: The molecular target of the FK506 neuroregenerative property following its application at a nerve injury and repair site, is still unknown. We investigated the role of FK506 binding proteins (FKBP)s, including FKBP12 and FKBP52, in the neuroregenerative action of locally applied FK506 using an in vitro three-dimensional compartmented cell culture system.
Method: The compartmented system consisted of a neonatal rat dorsal root ganglion (DRG) attached to the end of an acellular nerve allograft (ANA) that provided the native peripheral nerve scaffold (Fig.1). In the experimental groups, FK506 (100 ng/mL) was delivered exclusively to the growing neurites, the DRGs, or both in vitro. Such exclusive delivery was enabled by a silicone sheet that isolated the culture media surrounding the growing neurites and the DRG (Fig.1). The DRG-ANA constructs in the negative control group were not cultured with FK506. In a subset of samples in each group, the function of FKBP12 and/or FKBP52 was blocked by treating the DRG-ANA constructs with 100 ng/mL of antibodies for FKBP12 and/or FKBP52. Following 3 days of incubation at 37oC, the length and density of the extended neurites and Schwann Cell (SC) density in the ANA were measured in longitudinal histological sections of the constructs. The effect of local delivery of FK506 on neurite extension and SC proliferation with or without the blocking of FKBP12 and/or FKBP52 was evaluated.
Results: Local administration of FK506 to only the growing neurites significantly increased both neurite extension and neurite density in the ANA as compared with the negative control group. However, FK506 delivery to only the DRG or both the DRG and ANA significantly increased neurite extension without any effect on the neurite density as compared with the negative control group. A significantly higher SC density within the ANA was observed following administration of FK506 to only the growing neurites or the entire DRG-ANA construct. Blocking FKBP52, but not FKBP12 when FK506 was applied to the growing neurites, blocked its action of increasing neurite extension, neurite density, and SC density.
Conclusion: This in vitro study demonstrated that FKBP52 mediates the neuroregenerative effect of locally applied FK506 to the peripheral nerve. The findings provide new insights into the molecular and cellular mechanisms of the proregenerative action of FK506 on the peripheral nerve.
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