Foxq1 Promotes Alveolar Epithelial Cell Death through Tle1-mediated Inhibition of the NFκB Signaling Pathway.

Acute lung injury is a common respiratory disease characterized by diffuse alveolar injury and interstitial edema, as well as a hyperinflammatory response, lung cell damage and oxidative stress. Foxq1, a member of the FOX family of transcription factors, is expressed in various tissues, such as the lungs, liver, and kidneys, and contributes to various biological processes, such as stress, metabolism, cell cycle arrest, and aging-related apoptosis. However, the role of Foxq1 in acute lung injury is unknown. We constructed ex vivo and in vivo acute lung injury models by lipopolysaccharide tracheal perfusion of ICR mice and conditioned medium stimulation of injured MLE-12 cells. Foxq1 expression was increased, and its localization was altered in our acute lung injury model. In normal or injured MLE-12 cells, knockdown of Foxq1 promoted cell survival, and overexpression had the opposite effect. This regulatory effect was likely mediated by Tle1 and the NFκB/Bcl2/Bax signaling pathway. These data suggest a potential link between Foxq1 and acute lung injury, indicating that Foxq1 can be used as a biomarker for the diagnosis of acute lung injury. Targeted inhibition of Foxq1 expression could promote alveolar epithelial cell survival and may provide a strategy for mitigating acute lung injury.

Fidgetin interacting with microtubule end binding protein EB3 affects axonal regrowth in spinal cord injury.

Fidgetin, a microtubule-severing enzyme, regulates neurite outgrowth, axonal regeneration, and cell migration by trimming off the labile domain of microtubule polymers. Because maintenance of the microtubule labile domain is essential for axon initiation, elongation, and navigation, it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury. In this study, we constructed rat models of spinal cord injury and sciatic nerve injury. Compared with spinal cord injury, we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased, whereas fidgetin decreased after peripheral nerve injury. Depletion of fidgetin enhanced axon regeneration after spinal cord injury, whereas expression level of end binding protein 3 (EB3) markedly increased. Next, we performed RNA interference to knockdown EB3 or fidgetin. We found that deletion of EB3 did not change fidgetin expression. Conversely, deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3. Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules. Finally, we deleted EB3 and overexpressed fidgetin. We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3. When fidgetin was deleted, the labile portion of microtubules was elongated, and as a result the length of axons and number of axon branches were increased. These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury. Furthermore, they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules.

Kif15 deficiency contributes to depression-like behavior in mice.

Neuropsychiatric disorders have a high incidence worldwide. Kinesins, a family of microtubule-based molecular motor proteins, play essential roles in intracellular and axonal transport. Variants of kinesins have been found to be related to many diseases, including neurodevelopmental/neurodegenerative disorders. Kinesin-12 (also known as Kif15) was previously found to affect the frequency of both directional microtubule transports. However, whether Kif15 deficiency impacts mood in mice is yet to be investigated. In this study, we used the CRISPR/Cas9 method to obtain Kif15-/- mice. In behavioral tests, Kif15-/- female mice exhibited prominent depressive characteristics. Further studies showed that the expression of BDNF was significantly decreased in the frontal cortex, corpus callosum, and hippocampus of Kif15-/- mice, along with the upregulation of Interleukin-6 and Interleukin-1ß in the corpus callosum. In addition, the expression patterns of AnkG were notably changed in the developing brain of Kif15-/- mice. Based on our previous studies, we suggested that this appearance of altered AnkG was due to the maladjustment of the microtubule patterns induced by Kif15 deficiency. The distribution of PSD95 in neurites notably decreased after cultured neurons treated with the Kif15 inhibitor, but total PSD95 protein level was not impacted, which revealed that Kif15 may contribute to PSD95 transportation. This study suggested that Kif15 may serve as a potential target for future depression studies.

Promising application of a novel biomaterial, light chain of silk fibroin combined with NT3, in repairment of rat sciatic nerve defect injury.

Autologous nerve transplantation is the gold standard for treating peripheral nerve defects, but it is associated with defects such as insufficient donor and secondary injury. Artificial nerve guidance conduits (NGCs) are now considered promising alternatives for bridging long nerve gaps, although exploring new biomaterials to construct NGCs remains challenging. Silk fibroin (SF) has good biocompatibility and can self-assemble in aqueous solutions. However, the lack of proximal neurotrophic factors after nerve injury is a major concern, leading to incomplete nerve regeneration. In this study, NT-3, a neurotrophin that promotes neuronal survival and differentiation, was bound to the light chain of silk fibroin (FIBL) in two ways: one was directly bound to FIBL (FIBL-NT3) and the other was a polypeptides-linker (FIBL-Linker-NT3). The design aimed to take advantage of silk fiber's character of self-assembly of heavy-light chains and test whether a flexible linker with NT3 molecule is easy to be a NT3 dimer, the active form. In vitro studies indicated that FIBL-Linker-NT3 combined with SF membranes promoted axon growth in adult rat dorsal root ganglion (DRG) neurons. Then we tested if FIBL-Linker-NT3 could self-assemble with the SF heavy chain (SFH). DTT (Dithiothreitol) was used to break the disulfide bonds between the SF light and heavy chains, and the light-chain protein was removed via dialysis. SFH was assembled using FIBL-Linker-NT3, as evidenced by the western blotting results that showed a high molecular band corresponding to SFH-FIBL-Linker-NT3. Chitosan scaffolds have been identified to provide a suitable microenvironment, so a chitosan/SF-FIBL-Linker-NT3 conduit was also constructed. Nerve transplantation of this conduit was evaluated in vivo in a rat sciatic nerve defect model. Immunohistochemical assays showed that the chitosan/SF-FIBL-Linker-NT3 group was superior to the chitosan/PBS, SF, PBS + FIBL-Linker-NT3 groups in nerve regeneration. In addition, the chitosan/SF-FIBL-Linker-NT3 conduit-transplanted group exhibited better recovery in terms of neurite length, sciatic functional index value, sensitivity to heat, time on the rotarod, wet weight ratio, cross-sectional area, compound muscle action potential, number of myelin layers, and myelin thickness in the nerve. Taking together, our study identified that FIBL-Linker-NT3 could promote axonal growth and regeneration in vivo and in vitro and is a promising candidate biomaterial for artificial NGCs.

Deficiency of Kif15 gene inhibits tumor growth due to host CD8+T lymphocytes increase.

Kif15, also name kinesin-12, is a microtubule (MT) associate protein, which functions as a regulator of MT-dependent transport or spindle organization. Previous studies reported Kif15 increases in many tumors, however the effect of host Kif15 gene lack on tumor growth is not investigated. In this study, CRISPR/Cas9 mediated Kif15 gene knockout (Kif15-/-) mice were established and HE (Hematoxylin-Eosin) assay revealed no significant differences of morphology in most adult tissues (heart, liver, lung, kidney, and brain) except a retarded development of spleen in adult Kif15-/- mice. RNA sequence analysis of adult spleen tissues of Kif15-/- and Kif15+/+ mice was performed, and the results revealed that a total of 438 mRNAs were significantly differentially expressed in Kif15 knockout spleen, showing the top biological process was immune system process. FCM (Flow Cytometry) assay showed the percentage of CD8+ T lymphocytes notably increased in spleens of 9 w and 12 w old Kif15-/- mice. The CD8+ T lymphocytes are cytotoxic effector cells fighting against tumor. We thus detected the tumor growth in Kif15-/- mice using the melanoma cells inoculated subcutaneously. The tumor size significantly reduced in Kif15-/- mice. We finally detected whether Kif15 dysfunction affects the phagocytic function of macrophages on tumor cells, and the result showed Kif15 inhibitor treated macrophages significantly promoted the phagocytosis in vitro. In summary, this study revealed that the tumor-bearing mice of Kif15 gene deficiency notably inhibited tumor growth due to innate immune activation, which was the first report of the relation of Kif15 on the immunoreactivity.

Fidgetin impacts axonal growth and branching in a local mTOR signal dependent manner.

Neurons require a constant increase in protein synthesis during axonal growth and regeneration. AKT-mTOR is a central pathway for mammalian cell survival and regeneration. Fidgetin (Fign) is an ATP-dependent microtubule (MT)-severing enzyme whose functions are associated with neurite outgrowth, axon regeneration and cell migration. Although most previous studies have indicated that depletion of Fign is involved in those biological activities by increasing labile MT mass, it remains unknown whether mTOR activation contributes to this process. Here, we showed that depletion of Fign enhanced p-mTOR/p-S6K activation, and the mTOR inhibitor Rapamycin inhibited axon outgrowth and p-rpS6 activation. We then investigated the effects of neuronal-specific Fign deletion in a rat spinal cord hemisection model by injecting syn-GFP Fign shRNA virus. BBB values revealed an improvement in functional recovery. The p-mTOR was activated along with neuronal Fign depletion. The syn-mCherry virus showed more sprouting neurites entering the injury region, which was confirmed by immunostaining GAP43 protein. Further, we showed that Fign siRNA treatment promoted axon outgrowth and branching, whose underlying mechanism was firstly attributed to local activation of the mTOR pathway, and increased MT dynamicity. Finally, considering L-leucine, promotes axonal growth and neuronal survival, we applied L-leucine with Fign depletion after spinal cord injury or in chondroitin sulfate proteoglycan inhibitory molecules. The phenomenon of synergistically augmented axon regeneration was observed. In summary, our results indicated a novel local mTOR pathway for fidgetin to impact axon growth and provided a combined strategy in SCI.