Hsp90-binding immunophilins mediate neurodifferentiation and neuroregeneration

CRISTINA DANERI-BECERRA, MICHELLE PATIÑO, MARIO D. GALIGNIANA

Turku

Congreso; VIII International Congress on Stress Proteins in Biology & Medicine; 2017

Cell Stress Society International

Immunophilins are a family of intracellular proteins that possess peptidyl-prolyl isomerase enzymatic activity. They are also intracellular receptors for immunosuppressive drugs. Those immunophilins that bind the macrolide FK506 are referred to as FK506-binding proteins (or FKBPs), whereas those that bind the cyclic undecapeptide cyclosporine A (CsA) are referred to as cyclophilins (or CyPs). FKBP51 (51-kDa) and FKBP52 (52 kDa) are Hsp90-binding immunophilins belonging to the Immunophilin?Hsp90?Hsp70?p23 chaperone heterocomplex associated to steroid receptors. In previous studies, we demonstrated that FK506 shows both neuroregenerative and neuroprotective actions via FKBP immunophilins. Thus, neurite outgrowth is favoured by FKBP52 overexpression or FKBP51 knock-down, whereas it is impaired by FKBP52 knock-down or FKBP51 overexpression, suggesting that the expression balance between these two FK506-binding proteins plays a key role during neuronal differentiation. In this study, we aimed to demonstrate such neurotrophic action in vivo using murine models, and extend the analysis to CsA, the specific ligand of CyP immunophilins. Both drugs were able to promote neurite outgrowth in the undifferentiated N2a neuroblastoma cell line. Then, we performed organotypic cultures of 250 µm thick slices of prefrontal brain cortex or spinal cord. Assessment of cell death was performed using LDH, CPK and TGO enzymatic activity as markers of cell death at different times after culturing. Cultured slices were allowed to recovery for at least three days before performing treatments with 0.5 mM H2O2 and/or 1 µM FK506 or 1 µM CsA, alone or combined. Interestingly, drug pretreatment attenuated the induction of HSP70 and HSP90 observed by treatments with the peroxide, suggesting a protective action on the nervous tissue. Importantly, the subcellular fractionation of prefrontal brain slices showed that drug treatments preserves FKBP51 and HSP70 in mitochondria, which contrasts with the mitochondrial export of both chaperones after the onset of a deleterious stimulus such as H2O2. This is in agreement with previous studies where we demonstrated that both HSP70 and FKBP51 abandon mitochondria to preserve cells from apoptosis. Western blots show similar results after intracerebral injections of mice in the frontoparietal cortex of the right hemisphere with CoCl2 to generate a hypoxia-like condition (Co2+ replaces Fe2+ in the haeme chelate), and saline solution in the contralateral hemisphere as control. Importantly, in vivo responses were also greatly favoured by FK506 or CsA treatment. Thus, to detect motor impairments a Rotarod test was assessed. Again, FK506- or CsA-treated mice showed a better performance in parameters such as walked distance, speed of translation, and time of stability on the drum. In order to analyze the locomotor recovery of mice, a spinal cord full injury was performed under an operating microscope by transecting the spinal cord at thoracic level (T10). Mice were injected (s.c.) daily with 0.1 mg/Kg of FK506 or CsA, and a BMS (Basso Mouse Scale) test was performed. Locomotor recovery was significantly faster and greater in drug-treated mice than in vehicle-injected controls. Importantly, recovery was better in FKBP51-KO mice since this immunophilin is an inhibitory protein, whereas recovery was less efficient in FKBP52-KO mice since this immunophilin is a positive factor. In agreement with these results, microscopy studies of tissue for the expression of trophic factors or inflammatory reaction was greatly improved by treatments with the immunophilin ligands. In summary, this study proposes that treatments with immunophilin ligands could be beneficial for neurotrophic action.