Until recently, the expression of cathepsin K was believed to be limited to osteoclasts, where it serves a central function for the proper turnover of long bones [2–4]. Cathepsin K expression has since been shown in a variety of organs and tissues, and has been connected to numerous other functions, such as proper signalling in the context of the immune response  or the processing of β-endorphin in the brain . Cathepsin K has also been associated with diseases, such as breast cancer  and schizophrenia . Further functions of cathepsin K with a widespread impact on physiology and pathophysiology comprise its contributions in glucose metabolism  and in the processing of one of its natural substrates, thyroglobulin, thereby mediating liberation of thyroid hormones [8, 12, 13, 16]. Hence, more detailed investigations concerning other potential roles of this protease are essential, especially considering the entry of cathepsin K inhibitors into clinical treatment of osteoporosis [7, 8, 34]. The present study aimed to characterize possible functions of cathepsin K in the mouse CNS. Our results demonstrate that cathepsin K is of considerably higher importance for the development, structure, and function of the CNS than previously thought.
From the results of this study, we concluded that the metabolism and structure of non-neuronal cells were significantly perturbed in the CNS of Ctsk
-/- animals. The numbers and the maturation level of astrocytes were decreased in the hippocampus, while oligodendrocyte markers were altered in all analyzed brain regions except the cortex. The distribution of microglia was modified only in the cortex. The analysis of neuronal markers demonstrated that the architecture of the neuronal layers was affected by cathepsin K deficiency in particular in the hippocampus, a region of the CNS known for its importance in the regulation of anxiety and memory. In line with this notion, we observed the highest specific activity of cathepsin K within this brain region.
Finally, behavioral studies showed that general locomotor function was not obviously affected in Ctsk
-/- mice, at least up to the age of 6 months. However, importantly, a clear impact of cathepsin K deficiency on learning and memory as well as novelty seeking was revealed.
Linking brain and thyroid functions of cathepsin K
Previously, we have shown that cathepsin K- and L-double deficient mice are hypothyroid . Hypothyroid conditions in early post-natal stages are considered to negatively affect the development of the CNS . The abnormal state of cerebellar development during this period is one of the most obvious indications for severe hypothyroidism expected to affect adult brain structure and function significantly . However, cerebellar development of Ctsk
mice at post-natal day 12 was indistinguishable from that of WT controls inspected at the same age. Moreover, thyroxine levels in the blood of the Ctsk
-/- animals analyzed in our previous study and those investigated in this study were not significantly altered in comparison to WT controls. Thus, although we initially hypothesized that the brain phenotype described in this study could be directly attributed to a mild hypothyroidism, we concluded that this hypothesis was supported neither by the status of early post-natal development of Ctsk
mice nor by the thyroid hormone levels determined in the brain of these animals (Sîrbulescu, Dauth, Rehders, Saftig, Jordans, Brix, unpublished). Therefore, we addressed other mechanisms through which cathepsin K deficiency could have affected brain development. Because we show herein that cathepsin K is indeed expressed in the mouse brain and exhibits proteolytic activity in particular in the hippocampus, we suggest a more direct effect of cathepsin K deficiency on the mouse CNS development. To unequivocally distinguish between the effects exerted by a lack of cathepsin K expression in either the thyroid or the CNS of mice, organ-specific knock-outs are required and planned for in our future investigations.
Proteolytic network of the cathepsin K-deficient mouse CNS
Another pathway which might lead to the pronounced changes observed in the CNS of Ctsk
-/- mice is via modulation of related proteases. Compensatory phenomena underlying the mild phenotypes in mice lacking only one cysteine cathepsin have been previously described to occur in a cell type and tissue specific manner [1, 8]. Interestingly, while cathepsins B and L, which are closely related to cathepsin K, showed altered expression levels in various brain regions, the distantly related aspartic protease cathepsin D was not affected in the brain of Ctsk
Cathepsin B has been associated with neuronal survival by a variety of studies, often with conflicting results. While cathepsin B of microglial origin has been shown to serve as an initiator of apoptosis in cultures of hippocampal  and cortical neurons , its absence has been associated with the death of motor neurons in amyotrophic lateral sclerosis . A cathepsin B-like protease has been implicated in dendritic spine collapse in hippocampal neurons, a process which mediates neuronal synaptic plasticity . A decrease in protein level or activity of this protease, as observed in the cortex and striatum/mesencephalon of Ctsk
-/- mice, may therefore carry a multitude of consequences, including an overall enhancement of neuronal activity and/or metabolism. Moreover, the decrease in the activity levels of cathepsin B correlated with a strong increase in the levels of synaptotagmin in the striatum/mesencephalon, suggesting that mechanisms mediating neuronal plasticity may be altered in Ctsk
Cathepsin L, the most similar in structure and function to cathepsin K, showed a significant decrease in protein levels in the cortex of Ctsk
-/- mice, while in the hippocampus an increase was observed. Activity, however, was moderately decreased in the striatum/mesencephalon, hippocampus, and cerebellum. A series of studies suggest that cathepsin L is involved in the maturation of the peptide neurotransmitter enkephalin from its proenkephalin precursor . In turn, enkephalin has been shown to interact with the dopaminergic system via D2 receptors .
It is interesting to note that we have observed variations not only in the expression levels of cathepsins B and L, but also in those of cystatin C, an endogenous cathepsin inhibitor with extracellular functions . Cystatin C may act as a safe-guarding inhibitor of cysteine cathepsins after their release from damaged cells under challenging conditions that involve cell death . A recent study described cystatin C as having neuroprotective effects on murine primary cortical neurons and neuronal cell lines, by inducing autophagy and thereby improving clearance of long-lived proteins . Cystatin C is also a well established marker of brain tumours such as gliomas . The significance of cystatin C upregulation in the hippocampus of Ctsk
-/- mice, thus, needs to be further investigated.
Cystatin B, another endogenous cysteine cathepsin inhibitor with mainly intracellular safe-guarding tasks, has been shown previously to be required for normal CNS function. Patients with cystatin B mutations suffer from the Unverricht Lundborg syndrome, a disorder associated with epileptic seizures . Because the cathepsin K-deficient mice exhibited striking neurobehavioral phenotypes, although no signs of epilepsy were detectable, it will be important in the future to determine the levels of cysteine cathepsin inhibitors in the CNS of Ctsk
-/- mice in more detail. In an elegant approach aiming at the understanding of fine-tuning proteolytic activities at the genome level, mouse models of cystatin B deficiency have been crossed with cathepsin B- and/or L-deficient animals  but not yet with Ctsk
-/- mice. From the results of this study and those of Houseweart and colleagues it is, however, clear that altered ratios of cysteine proteases and their inhibitors occur in the brain of cysteine cathepsin and/or cystatin-deficient mice. This is of relevance, considering that certain inherited mutations or an imbalance of cathepsins and their endogenous inhibitors in the brain of both mice and humans have been linked to a number of severe impairments [18, 19, 46].
Changes in cellular architecture resulting from cathepsin K deficiency
In the present work, we observed significantly fewer astrocytes in the hippocampus and significantly more in the cortex of Ctsk
-/- mice. Astrocytes have a wide range of functions that are highly relevant for CNS homeostasis and for proper neuronal function [47, 48]. In pathological situations, including neurodegeneration, activation of astrocytes as characterized by an overexpression of GFAP , and astrogliosis, an abnormal increase in the number of astrocytes in damaged areas of the CNS, has been observed . It is therefore not surprising that due to changes in maturation and numbers of astrocytes in Ctsk
-/- mice, other cell types like oligodendrocytes and neurons may be affected as well. An enhanced signal of the microglial marker Iba1 was detected in parallel to the increased astrocyte population in the cortex of Ctsk
-/- animals. This is interesting to note, because microglia respond to any kind of injury or perturbation of the CNS and it is believed that they play an essential role both in acute neuroinflammation and in wound healing [28, 50]. Therefore, higher levels of cortical Iba1 are in line with the observations of higher GFAP levels in the cortex of Ctsk
-/- mice, thereby putatively hinting to an altered composition of neurosupportive, neurotrophic, and repair-promoting factors in some regions of the brain.
Dopaminergic system of Ctsk
-/- mice and impact on neurobehavioral tasks
The overall changes observed in the dopaminergic system of Ctsk
-/- mice are of particular interest, since dopamine-mediated neuromodulation is involved in a wide variety of processes, including movement and motor learning, novelty seeking, reward processing, fear conditioning, food intake, nociception, and endocrine and autonomic regulation [29, 51, 52]. In addition, the dopaminergic system is known to have an important role in both spatial and non-spatial learning . Ctsk
-/- mice in the present study displayed significantly increased levels of tyrosine hydroxylase, the initial and rate-limiting enzyme in dopamine biosynthesis  and slightly increased levels of dopamine. Interestingly, D2-receptor levels were also significantly increased in the striatum/mesencephalon fraction of Ctsk
-/- mice. Agonists of D2 receptors, such as RU 24213 or quinpirole have been shown to induce learning impairments  and to decrease anxiety levels in mice , respectively. In line with this notion, in the present study we observed that Ctsk
-/- mice displayed reduced anxiety levels and impairments in learning and memory skills while exhibiting enhanced dopamine and increased D2-receptor levels in comparison to WT controls.
Another mechanism by which cathepsin K might affect the dopaminergic system could be via its contribution to opioid metabolism. Up-regulation of cathepsin K expression in rats was directly linked to schizophrenia induced by treatment with neuroleptics . Interestingly, post-mortem brain tissue from patients with schizophrenia also revealed an increase in the cathepsin K protein levels . A recent in vitro study has further shown that cathepsin K can metabolize β-endorphin to produce met-enkephalin . Met-enkephalin binds to opiate receptors, leading to an inhibitory effect on dopaminergic neurons of the VTA . Accordingly, we propose that in the absence of cathepsin K activity, less met-enkephalin would be produced, thus lowering the inhibitory effect on the VTA dopaminergic neurons and leading to increased dopaminergic output.
The deregulated cysteine cathepsin network, the alterations in the cellular architecture of the hippocampus, and the marked metabolic changes observed in Ctsk
-/- mice which included the dopaminergic system, prompted us to investigate whether these modifications resulted in altered behavioral patterns. Since both the hippocampus and the dopaminergic system are crucially involved in learning and memory  as well as in anxiety-related behavior , we focused on several tests addressing these aspects.
It is important to mention that, although cathepsin K is well known for its implication in bone remodeling [4, 7, 58], the mice used in this study did not show any sign of physical impairment, likely because they were used at a young age, of up to 6 months. Moreover, a pilot study on a separate group of Ctsk
-/- and WT animals (n = 10 for each genotype), using the pole test and the wire hang test, which assess motor abilities in mice , showed no difference between the two groups [Sîrbulescu, Jordans, Lerchl, Saftig, Brix; unpublished observations].
In a series of tests chosen to assess anxiety, exploration and memory [60–62], Ctsk
-/- mice showed decreased anxiety and marked spatial and non-spatial learning and memory impairments. Previous studies have linked an overactive dopaminergic system to reduced inhibition in rodents [51, 63]. Our results of the behavioral tests are therefore in line with the observed increase of dopaminergic output in Ctsk
-/- mice. Moreover, the neuronal cytoarchitecture of the ventral hippocampus was affected more severely by cathepsin K deficiency than that of the dorsal hippocampus. The ventral hippocampus has been shown to be involved in regulating emotion-related behavior . Lesions applied in this area typically lead to reduced anxiety levels in rodents . In addition, the alterations in astrocyte numbers and maturation states observed in the ventral hippocampus of Ctsk
animals may contribute to the observed changes in the distribution and numbers of neurons, which in turn can lead to the observed behavioral phenotypes.
mice also showed marked impairments in both non-spatial learning, as indicated by their inability to discriminate the introduction of a novel object into a familiar environment, and in spatial learning tasks, as demonstrated by the multiple choice maze paradigm. These results are in agreement with the profound metabolic disruptions found at the level of both the dopaminergic system and the hippocampus in these animals, because the dopaminergic system is known to have an important role in spatial and non-spatial learning  while the hippocampus is well established as a structure with an essential role for spatial but also for non-spatial memory formation .