Decreased turnover of the CNS myelin protein Opalin in a mouse model of hereditary spastic paraplegia 35.

Spastic paraplegia 35 (SPG35) (OMIM: 612319) or fatty acid hydroxylase-associated neurodegeneration (FAHN) is caused by deficiency of fatty acid 2-hydroxylase (FA2H). This enzyme synthesizes sphingolipids containing 2-hydroxylated fatty acids, which are particularly abundant in myelin. Fa2h-deficient (Fa2h-/-) mice develop symptoms reminiscent of the human disease and therefore serve as animal model of SPG35. In order to understand further the pathogenesis of SPG35, we compared the proteome of purified CNS myelin isolated from wild type and Fa2h-/- mice at different time points of disease progression using tandem mass tag labeling. Data analysis with a focus on myelin membrane proteins revealed a significant increase of the oligodendrocytic myelin paranodal and inner loop protein (Opalin) in Fa2h-/- mice, whereas the concentration of other major myelin proteins was not significantly changed. Western blot analysis revealed an almost 6-fold increase of Opalin in myelin of Fa2h-/- mice aged 21-23 months. A concurrent unaltered Opalin gene expression suggested a decreased turnover of the Opalin protein in Fa2h-/- mice. Supporting this hypothesis, Opalin protein half-life was reduced significantly when expressed in CHO cells synthesizing 2-hydroxylated sulfatide, compared to cells synthesizing only non-hydroxylated sulfatide. Degradation of Opalin was inhibited by inhibitors of lysosomal degradation but unaffected by proteasome inhibitors. Taken together, these results reveal a new function of 2-hydroxylated sphingolipids namely affecting the turnover of a myelin membrane protein. This may play a role in the pathogenesis of SPG35.

The PI3K inhibitor pictilisib and the multikinase inhibitors pazopanib and sorafenib have an impact on Rac1 level and migration of medulloblastoma in vitro.

Metastatic disease is the leading cause of death in children suffering from medulloblastoma and a major treatment challenge. The evidence of leptomeningeal dissemination defines the most aggressive tumours and is associated with increased mortality; thus, inhibition of migration as a factor involved in the process of metastatic disease is fundamental for the treatment and prevention of metastatic dissemination. Targeting the small Rho GTPases Rac1 has been shown to effectively impair medulloblastoma cell migration in vitro. Yet clinically applicable selective Rac1 inhibitors are still lacking. In view of the pertinent oncogenic role of the PI3K signalling cascade and tyrosine kinase-mediated signalling pathways in medulloblastoma, we explored clinically available targeted therapeutics to this effect. Here, we show that Rac1 is expressed in both the cytoplasm and nucleus in the medulloblastoma cell lines Daoy and MEB-Med-8A representative of two high risk medulloblastoma entities. We demonstrate that activated Rac1 is subject to substantial downmodulation following administration of the clinically available inhibitor of the PI3K pathway Pictilisib (GDC-0941) and the multityrosine kinase inhibitors Pazopanib and Sorafenib. The application of those drugs was associated with reduced mobility of the medulloblastoma cells and alterations of the actin skeleton. Of note, PI3K inhibition reveals the strongest anti-migratory effect in Daoy cells. Thus, our in vitro observations provide new insights into different strategies of blocking Rac1 and inhibiting migration in medulloblastoma employing clinically available agents paving the way for confirmatory studies in in vivo models.

Age-Dependent Increase in Schmidt-Lanterman Incisures and a Cadm4-Associated Membrane Skeletal Complex in Fatty Acid 2-hydroxylase Deficient Mice: a Mouse Model of Spastic Paraplegia SPG35.

PNS and CNS myelin contain large amounts of galactocerebroside and sulfatide with 2-hydroxylated fatty acids. The underlying hydroxylation reaction is catalyzed by fatty acid 2-hydroxylase (FA2H). Deficiency in this enzyme causes a complicated hereditary spastic paraplegia, SPG35, which is associated with leukodystrophy. Mass spectrometry-based proteomics of purified myelin isolated from sciatic nerves of Fa2h-deficient (Fa2h-/-) mice revealed an increase in the concentration of the three proteins Cadm4, Mpp6 (Pals2), and protein band 4.1G (Epb41l2) in 17-month-old, but not in young (4 to 6-month-old), Fa2h-/- mice. These proteins are known to form a complex, together with the protein Lin7, in Schmidt-Lanterman incisures (SLIs). Accordingly, the number of SLIs was significantly increased in 17-month-old but not 4-month-old Fa2h-/- mice compared to age-matched wild-type mice. On the other hand, the relative increase in the SLI frequency was less pronounced than expected from Cadm4, Lin7, Mpp6 (Pals2), and band 4.1G (Epb41l2) protein levels. This suggests that the latter not only reflect the higher SLI frequency but that the concentration of the Cadm4 containing complex itself is increased in the SLIs or compact myelin of Fa2h-/- mice and may potentially play a role in the pathogenesis of the disease. The proteome data are available via ProteomeXchange with identifier PXD030244.

Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits.

BACKGROUND: Cathepsin 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. RESULTS: Cathepsin 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. CONCLUSION: We 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.

Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions.

BACKGROUND: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. RESULTS: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. CONCLUSION: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.

Cysteine cathepsins: cellular roadmap to different functions.

Cysteine cathepsins belong to the papain-like family C1 of clan CA cysteine peptidases. These enzymes are ubiquitously expressed and exert their proteolytic activity mainly, but not exclusively within the compartments along the endocytic pathway. Moreover, cysteine cathepsins are active in pericellular environments as soluble enzymes or bound to cell surface receptors at the plasma membrane, and possibly even within secretory vesicles, the cytosol, mitochondria, and within the nuclei of eukaryotic cells. Proteolytic actions performed by cysteine cathepsins are essential in the maintenance of homeostasis and depend heavily upon their correct sorting and trafficking within cells. As a consequence, the numerous and diverse approaches to identification, qualitative and quantitative determination, and visualization of cysteine cathepsin functions in vitro, in situ, and in vivo cover the entire spectrum of biochemistry, molecular and cell biology. This review focuses upon the transport pathways directing cysteine cathepsins to their points of action and thus emphasizes the broader role and functionality of cysteine cathepsins in a number of specific cellular locales. Such understanding will provide a foundation for future research investigating the involvement of these peptidases with their substrates, inhibitors, and the intertwined proteolytic networks at the hubs of complex biological systems.

Cathepsin B trafficking in thyroid carcinoma cells.

BACKGROUND: The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in prohormone processing initiated in the follicle lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space in thyroid cancer tissue, and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through e.g. extracellular matrix degradation. METHODS: Transport of cathepsin B in normal thyroid epithelial and carcinoma cells was investigated through immunolocalization of endogenous cathepsin B in combination with probing protease activity. Transport analyses of cathepsin B-eGFP and its active-site mutant counterpart cathepsin B-C29A-eGFP were used to test whether intrinsic sequences of a protease influence its trafficking. RESULTS: Our approach employing activity based probes, which distinguish between active and inactive cysteine proteases, demonstrated that both eGFP-tagged normal and active-site mutated cathepsin B chimeras reached the endo-lysosomal compartments of thyroid epithelial cells, thereby ruling out alterations of sorting signals by mutagenesis of the active-site cysteine. Analysis of chimeric protein trafficking further showed that GFP-tagged cathepsin B was transported to the expected compartments, i.e. endoplasmic reticulum, Golgi apparatus and endo-lysosomes of normal and thyroid carcinoma cell lines. However, the active-site mutated cathepsin B chimera was mostly retained in the endoplasmic reticulum and Golgi of KTC-1 and HTh7 cells. Hence the latter, as the least polarized of the three carcinoma cell lines analyzed, exhibited severe transport defects in that it retained chimeras in pre-endolysosomal compartments. Furthermore, secretion of endogenous cathepsin B and of other cysteine peptidases, which occurs at the apical pole of normal thyroid epithelial cells, was most prominent and occurred in a non-directed fashion in thyroid carcinoma cells. CONCLUSIONS: Transport of endogenous and eGFP-tagged active and inactive cathepsin B in the cultured thyroid carcinoma cells reflected the distribution patterns of this protease in thyroid carcinoma tissue. Hence, our studies showed that sub-cellular localization of proteolysis is a crucial step in regulation of tissue homeostasis. We conclude that any interference with protease trafficking resulting in altered regulation of proteolytic events leads to, or is a consequence of the onset and progression of thyroid cancer.

Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells.

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.