Peripheral neuropathy is accompanied by changes in the neuronal environment. The blood-nerve barrier (BNB) is crucial in protecting the neural homeostasis: Tight junctions (TJ) seal paracellular spaces and thus prevent external stimuli from entering. In different models of neuropathic pain, the BNB is impaired, thus contributing to local damage, immune cell invasion and, ultimately, the development of neuropathy with its symptoms. In this study, we examined changes in expression and microstructural localization of two key tight junction proteins (TJP), claudin-1 and the cytoplasmic anchoring ZO-1, in the sciatic nerve of mice subjected to chronic constriction injury (CCI). Via qPCR and analysis of fluorescence immunohistochemistry, a marked downregulation of mRNA as well as decreased fluorescence intensity were observed in the nerve for both proteins. Moreover, a distinct zig-zag structure for both proteins located at cell-cell contacts, indicative of the localization of TJs, was observed in the perineurial compartment of sham-operated animals. This microstructural location in cell-cell-contacts was lost in neuropathy as semiquantified via computational analysis, based on a novel algorithm. In summary, we provide evidence that peripheral neuropathy is not only associated with decrease in relevant TJPs but also exhibits alterations in TJP arrangement and loss in barrier tightness, presumably due to internalization. Specifically, semiquantification of TJP in cell-cell-contacts of microcompartments could be used in the future for routine clinical samples of patients with neuropathy.

Running title: Loss of claudin-12 results in mechanical hypersensitivity. AbstractPeripheral nerves and their axons are shielded by the blood-nerve and the myelin barrier, but understanding of how these barriers impact nociception is limited. Here, we identified a regulatory axis of the tight junction protein claudin-12, sex-dependently controlling perineurial and myelin barrier integrity. In nerve biopsies, claudin-12 in Schwann cells was lost in male and postmenopausal female patients with painful but not painless polyneuropathy. Global Cldn12 gene-knockout selectively increased perineurial/myelin barrier leakage, damaged tight junction protein expression and morphology, increased proinflammatory cytokines and induced mechanical hypersensitivity in naïve and neuropathic male mice, respectively. Other barriers and neurological function remained intact. In vitro transfection studies documented claudin-12 plasma membrane localisation without interaction with other tight junction proteins or intrinsic sealing properties. Rather, claudin-12 had a regulatory tight junction protein function on the myelin barrier via the morphogen SHH in vivo in Cldn12-KO and after local siRNA knockdown. Fertile female mice were completely protected. Collectively, these studies reveal the critical role of claudin-12 maintaining the myelin barrier and highlight restoration of the claudin-12/SHH pathway as a potential target for painful neuropathy. 5

No abstract