The asymmetric molecular forms of AChE and the expression of collagen Q in mature and immature fast and slow rat muscles.

There is a major difference between fast and slow rat muscles in regard to acetylcholinesterase (AChE) expression in their extrajunctional regions: the activity of the asymmetric forms of AChE (A(8) and A(12)) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the nearly complete suppression of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. This difference is partly caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the levels of mRNA of ColQ. Whereas the changes of the levels of ColQ mRNA in slow muscles, observed in response to different electrical stimulation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible in this respect. Calcineurin signaling pathway in slow muscle fibers, activated by high average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, maintains high mRNA levels of ColQ in the extrajunctional regions of the slow soleus muscles. A different, calcineurin-independent regulatory pathway is responsible for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers. Immature rat muscle fibers, both fast and slow, however, display relatively high levels of the A forms of AChE and ColQ mRNA during the early postnatal period. Four days after birth, ColQ mRNA levels are already 2-fold higher in slow than in fast muscle fibers. Muscle regeneration after injury is a repetition of its ontogenetic development, originating from the muscle satellite cells. The extrajunctional levels of ColQ mRNA in non-innervated regenerating fast and slow muscles, however, are not significantly different, but they become about 2- to 3-fold higher in the regenerating soleus than in the fast STM already after several days of innervation by their respective nerves. We are currently testing a hypothesis that intrinsic differences exist between fast and slow muscle fibers in regard to their capacity to express ColQ extrajunctionally, and that these differences may originate in the stem cells of these muscle fibers.

Toward understanding recurrent meningioma: the potential role of lysosomal cysteine proteases and their inhibitors.

OBJECT: The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. METHODS: Primary meningioma cultured spheroids were "confronted" with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. RESULTS: The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). CONCLUSIONS: The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles--the role of calcineurin signaling.

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A(8) and A(12)) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.

Neural regulation of acetylcholinesterase-associated collagen Q in rat skeletal muscles.

Acetylcholinesterase-associated collagen Q is expressed also outside of neuromuscular junctions in the slow soleus muscle, but not in fast muscles. We examined the nerve dependence of muscle collagen Q expression and mechanisms responsible for these differences. Denervation decreased extrajunctional collagen Q mRNA levels in the soleus muscles and junctional levels in fast sternomastoid muscles to about one third. Cross-innervation of denervated soleus muscles by a fast muscle nerve, or electrical stimulation by 'fast' impulse pattern, reduced their extrajunctional collagen Q mRNA levels by 70-80%. In contrast, stimulation of fast muscles by 'slow' impulse pattern had no effect on collagen Q expression. Calcineurin inhibitors tacrolimus and cyclosporin A decreased collagen Q mRNA levels in the soleus muscles to about 35%, but did not affect collagen Q expression in denervated soleus muscles or the junctional expression in fast muscles. Therefore, high extrajunctional expression of collagen Q in the soleus muscle is maintained by its tonic nerve-induced activation pattern via the activated Ca(2+)-calcineurin signaling pathway. The extrajunctional collagen Q expression in fast muscles is independent of muscle activation pattern and seems irreversibly suppressed. The junctional expression of collagen Q in fast muscles is partly nerve-dependent, but does not encompass the Ca(2+)-calcineurin signaling pathway.

The role of muscle activation pattern and calcineurin in acetylcholinesterase regulation in rat skeletal muscles.

Acetylcholinesterase (AChE) expression in fast rat muscles is approximately fourfold higher than in slow muscles. We examined whether different muscle activation patterns are responsible for this difference and whether the calcineurin signaling pathway is involved in AChE regulation. The slow soleus and fast extensor digitorum longus (EDL) muscles were directly or indirectly stimulated by a tonic low-frequency or a phasic high-frequency pattern of electric impulses. The phasic, but not tonic, stimulation increased the AChE mRNA levels in denervated soleus muscles to those in the normal EDL and maintained high levels of AChE mRNA in denervated EDL muscles. Therefore, muscle activation pattern is the predominant regulator of extrajunctional AChE expression in rat muscles. Indirect phasic stimulation of innervated muscles, imposed on their natural pattern of neural activation, did not increase the AChE transcript levels in the soleus, whereas a 30% reduction was observed in the EDL muscles. A low number of impulses per day is therefore prerequisite for high AChE expression. Treatment by tacrolimus and cyclosporin A, two inhibitors of calcineurin (but not by a related substance rapamycin, which does not inhibit calcineurin), increased the levels of AChE transcripts in the control soleus muscles and in tonically electrically stimulated soleus and EDL muscles, even to reach those in the control EDL muscles. Therefore, tonic muscle activation reduces the extrajunctional levels of AChE transcripts by activating the calcineurin signaling pathway. In denervated soleus and EDL muscles, tacrolimus did not prevent the reduction of AChE mRNA levels, indicating that a calcineurin-independent suppressive mechanism was involved.

Cathepsin L in glioma progression: comparison with cathepsin B.

OBJECTIVE: Lysosomal cysteine cathepsins have been implicated in tumor progression. This study is aimed to reveal differential expression and compare the prognostic significance of cathepsins B and L in glioma patients. METHODS: The histological slides of 82 patients with primary astrocytic tumors were reviewed. We evaluated the immunostaining of the cathepsins in tumor and endothelial cells. RESULTS: Cathepsins B and L stained positive in 98 and 88% of cases, respectively. The total score was significantly higher in malignant than in benign tumors, both for cathepsin B (p<0.001) and for cathepsin L (p<0.01). The IHC score in endothelial cells in the malignant group was significantly higher only for cathepsin B (p<0.0001). Survival analysis indicated that in contrast to the prognostic significance of total cathepsin B and endothelial cells associated cathepsin B for shorter patients' survival, the prognostic role of cathepsin L was not confirmed. CONCLUSION: Cathepsin L is preferentially expressed in tumor cells, increasing with glioma progression, but is not significantly associated with new vasculature of glioblastoma. In contrast to cathepsin B, cathepsin L has no prognostic impact, suggesting different roles of the two cathepsins in glioma progression.