Cathepsin B but not cathepsins L or S contributes to the pathogenesis of Unverricht‐Lundborg progressive myoclonus epilepsy (EPM1)

Abstract

The inherited epilepsy Unverricht-Lundborg disease (EPM1) is caused by loss-of-function mutations in the cysteine protease inhibitor, cystatin B. Because cystatin B inhibits a class of lysosomal cysteine proteases called cathepsins, we hypothesized that increased proteolysis by one or more of these cathepsins is likely to be responsible for the seizure, ataxia, and neuronal apoptosis phenotypes characteristic of EPM1. To test this hypothesis and to identify which cysteine cathepsins contribute to EPM1, we have genetically removed three candidate cathepsins from cystatin B-deficient mice and tested for rescue of their EPM1 phenotypes. Whereas removal of cathepsins L or S from cystatin B-deficient mice did not ameliorate any aspect of the EPM1 phenotype, removal of cathepsin B resulted in a 36-89% reduction in the amount of cerebellar granule cell apoptosis depending on mouse age. The incidence of an incompletely penetrant eye phenotype was also reduced upon removal of cathepsin B. Because the apoptosis and eye phenotypes were not abolished completely and the ataxia and seizure phenotypes experienced by cystatin B-deficient animals were not diminished, this suggests that another molecule besides cathepsin B is also responsible for the pathogenesis, or that another molecule can partially compensate for cathepsin B function. These findings establish cathepsin B as a contributor to the apoptotic phenotype of cystatin B-deficient mice and humans with EPM1. They also suggest that the identification of cathepsin B substrates may further reveal the molecular basis for EPM1.

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

“…This view is strongly supported by the decreased cerebellar granule cell apoptosis detected in mice double-deficient for CSTB and cathepsin B. 18 Yet, the severity of the ataxia and seizure phenotypes were not reduced, 18 suggesting that CSTB might have other functions than cathepsin inhibition. The localization of CSTB both in the cytoplasm and nucleus allows one to speculate a role for CSTB in signalling events involved in apoptosis with downstream effects of transcriptional regulation.…”

“…17 In mice double-deficient for CSTB and cathepsin B, the amount of cerebellar apoptosis is significantly decreased, suggesting that cathepsin B contributes to the apoptotic phenotype in EPM1. 18 Moreover, seizures induce upregulation of CSTB mRNA and protein in rats. 19 Together, these findings strongly implicate a role for CSTB in neuroprotection.…”

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

“…There is increasing evidence that the ubiquitous lysosomal cysteine protease, catB, plays a specific role in a number of cellular processes, including the onset of acute pancreatitis (Halengk et al, 2000), the pathogenicity of myoclonus epilepsy (Houseweart et al, 2003), and TNF-a-induced hepatocyte apoptosis (Guicciardi et al, 2001). Our in vitro studies suggest that the induction of catB gene expression in differentiating myoblasts involves the MyoD family of skeletal-muscle specific transcription factors binding to E-box promoter elements of the catB gene promoter (Jane et al, 2002c), and that the resultant catB activity plays a specific role in myogenesis in vivo, most likely in the events associated with myoblast-myoblast fusion (Jane et al, 2002a,b).…”

Apoptosis caused by cathepsins does not require Bid signaling in an in vivo model of progressive myoclonus epilepsy (EPM1)

“…29 For this reason, we used the cystatin B-deficient mouse system to determine if in vivo proapoptotic signaling by cathepsins requires the presence of Bid to initiate or propagate apoptosis. Our initial test of this proposed mechanism was to compare amounts of Bid cleavage in wild-type and cystatin B-deficient cerebella.…”

“…The in vivo system we selected was a murine model of inherited epilepsy in which cathepsins contribute to the initiation or propagation of apoptosis when their endogenous cysteine protease inhibitor, cystatin B, is missing. 29 These cystatin B-deficient mice experience widespread cerebellar granule cell apoptosis, ataxia, and seizures, just as do humans with UnverrichtLundborg progressive myoclonus epilepsy (EPM1) who lack cystatin B. 30 With the endogenous inhibitor cystatin B missing, cathepsins are free to participate in activating the apoptotic pathway, and thereby contribute to the pathogenesis of EPM1 disease.…”