BackgroundCathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS). Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant.ResultsCathepsin K messenger RNA was detectable in all brain regions of wild type (WT) mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk-/-) animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk-/- mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of the dopaminergic system was found in Ctsk-/- animals which exhibited reduced anxiety levels as well as short- and long-term memory impairments in behavioral assessments.ConclusionWe conclude that deletion of the Ctsk gene can lead to deregulation of related proteases, resulting in a wide range of molecular and cellular changes in the CNS with severe consequences for tissue homeostasis. We propose that cathepsin K activity has an important impact on the development and maintenance of the CNS in mice.

Proteases play essential roles in protein degradation, protein processing, and extracellular matrix remodeling in all cell types and tissues. They are also involved in protein turnover for maintenance of homeostasis and protein activation or inactivation for cell signaling. Proteases range in function and specificity, with some performing distinct substrate cleavages, while others accomplish proteolysis of a wide range of substrates. As such, different cell types use specialized molecular mechanisms to regulate the localization of proteases and their function within the compartments to which they are destined. Here, we focus on the cysteine family of cathepsin proteases and legumain, which act predominately within the endo-lysosomal pathway. In particular, recent knowledge on cysteine cathepsins and their primary regulator legumain is scrutinized in terms of their trafficking to endo-lysosomal compartments and other less recognized cellular locations. We further explore the mechanisms that regulate these processes and point to pathological cases which arise from detours taken by these proteases. Moreover, the emerging biological roles of specific forms and variants of cysteine cathepsins and legumain are discussed. These may be decisive, pathogenic, or even deadly when localizing to unusual cellular compartments in their enzymatically active form, because they may exert unexpected effects by alternative substrate cleavage. Hence, we propose future perspectives for addressing the actions of cysteine cathepsins and legumain as well as their specific forms and variants. The increasing knowledge in non-canonical aspects of cysteine cathepsin- and legumain-mediated proteolysis may prove valuable for developing new strategies to utilize these versatile proteases in therapeutic approaches.

No abstract

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

This mini-review asks how self-regulation of the thyroid gland is realized at the cellular and molecular levels by canonical and non-canonical means. Canonical pathways of thyroid regulation comprise thyroid stimulating hormone-triggered receptor signaling. As part of non-canonical regulation, we hypothesized an interplay between protease-mediated thyroglobulin processing and thyroid hormone release into the circulation by means of thyroid hormone transporters like Mct8. We proposed a sensing mechanism by different thyroid hormone transporters, present in specific subcellular locations of thyroid epithelial cells, selectively monitoring individual steps of thyroglobulin processing, and thus, the cellular thyroid hormone status. Indeed, we found that proteases and thyroid hormone transporters are functionally inter-connected, however, in a counter-intuitive manner fostering self-thyrotoxicity in particular in Mct8- and/or Mct10-deficient mice. Furthermore, the possible role of the G protein-coupled receptor Taar1 is discussed, because we detected Taar1 at cilia of the apical plasma membrane of thyrocytes in vitro and in situ. Eventually, through pheno-typing Taar1-deficient mice, we identified a co-regulatory role of Taar1 and the thyroid stimulating hormone receptors. Recently, we showed that inhibition of thyroglobulin-processing enzymes results in disappearance of cilia from the apical pole of thyrocytes, while Taar1 is re-located to the endoplasmic reticulum. This pathway features a connection between thyrotropin-stimulated secretion of proteases into the thyroid follicle lumen and substrate-mediated self-assisted control of initially peri-cellular thyroglobulin processing, before its reinternalization by endocytosis, followed by extensive endo-lysosomal liberation of thyroid hormones, which are then released from thyroid follicles by means of thyroid hormone transporters.