In vitro neuroprotection by either extracellular or endogenouse human CysC.

<p><b>A.</b> Light microscopy pictures of N2a cells incubated for 48 hours in medium containing serum or in serum-free medium in the absence or presence of different concentrations of CysC. Scale bar represents 100 µm. <b>B.</b> Neuronal survival was measured by counting live cells, and expressed as percentage of neuronal survival in cultures incubated in serum-containing medium. Data are the mean and SEM of 4 experiments. <b>C. D.</b> Primary rat cortical neurons were cultured in neurobasal medium in the presence (<b>C</b>) or absence (<b>D</b>) of B27-supplement and different concentrations of human CysC for 24 hours. Cell survival analyzed by the MTS assay is expressed as percentage of live cells in cultures incubated in B27-supplemented medium without CysC. Data are the mean and SEM of 3 experiments. F and P values determined by one way ANOVA for (C) were 85.09 and <0.0001 and for (D) 34.00 and <0.0001. <b>E</b> Primary cortical neurons isolated from brains of CysC knockout (CysCko), transgenic mice overexpressing human CysC (CysCtg), or wild type (WT) mice were incubated in B27-supplement containing or lacking media. Survival is expressed as percentage of live cells in wild type cultures incubated in B27-supplemented medium. For groups incubated with B27 the F and P values determined by one way ANOVA were 20.60 and 0.0007 and for groups incubated without B27 were 68.93 and <0.0001.</p

CysC does not protect neuronal cells from toxicity when autophagy is inhibited.

<p><b>A.</b> N2a cells were incubated for 44 hours in serum-free medium with 3MA, an inhibitor of autophagy, in the presence or absence of 0.4 µM CysC. Neuronal survival was measured by counting live cells, and expressed as percentage of neuronal survival in cultures incubated in serum-deprived medium. Data are the mean and SEM of 4 experiments. There were no significant differences between samples containing 3MA with or without CysC. <b>B.</b> N2a cells were treated with two forms of <i>beclin 1</i> siRNA (a and b). Western blot analysis with anti-beclin 1 antibody shows inhibition of beclin 1 expression in treated cells. <b>C. </b><i>Beclin 1</i> siRNA treatment of N2a cells attenuates the protective effect of CysC from serum-deprivation induced death. Cell survival was measured by the MTS assay. Data are the mean and SEM of 3 experiments. No significant difference between CysC treated and non- treated cells was observed.</p

CysC enhances total lysosomal-dependent protein degradation in serum-deprived neuronal cells.

<p><b>A.</b> Effect of increasing concentrations of CysC on total rates of protein degradation. N2a cells were labeled for 2 days with [³H]-leucine. After extensive washing, cells were incubated in serum-containing or serum-free media. Removal of serum maximally activates lysosomal degradation. The cells maintained in serum-free media were supplemented or not with increasing concentrations of CysC as labeled. The rate of total protein degradation at the indicated times was calculated as the percentage of total radiolabeled protein transformed into soluble amino acids. <b>B.</b> Effect of inhibition of lysosomal proteolysis on the CysC-induced increase in protein degradation. N2a cells were labeled as in A and then maintained in serum-free media and supplemented or not with CysC. Where indicated 20 mM NH₄Cl and 100 µM leupeptin were added to inhibit lysosomal proteolysis. Protein degradation was calculated as in A. <b>C</b>. Effect of CysC on macroautophagy-dependent proteolysis. N2a cells labeled as in A and maintained in serum-free media were supplemented or not with CysC. Half of the cells were treated with 10 mM 3MA to inhibit macroautophagy. The percentage of lysosomal degradation that results from autophagic degradation (3MA sensitive), in the presence or absence of CysC was calculated. Values are mean and SED of triplicate wells in 3–4 different experiments. One way ANOVA for differences between CysC treated and untreated samples were significant for *<i>p</i> = 0.05; **<i>p</i> = 0.001 and between control and ammonium chloride treated samples were significant for <b>⁺</b><i>p</i> = 0.01.</p

CysC induces an increase in the number of autophagic vesicles (AVs) in neuronal cells.

<p>Electron micrographs of primary rat cortical neurons (<b>A</b>) incubated for 12 hours in either B27-supplemented or deprived medium in the absence or presence of 0.75 µM CysC. The total number of AVs <i>per</i> cell was counted for at least 20 cells/condition, and the average number of vesicles <i>per</i> cell is shown (<b>B</b>). Data are the mean and SEM. Differences between CysC treated and untreated samples were calculated by Student's t-test.</p

CysC induces autophagy via the mTOR signaling pathway in either serum-containing or serum-free medium.

<p><b>A.</b> N2a cells were incubated for 12 hrs in serum-containing or serum-free medium in the presence or absence of CysC. Cell lysate proteins were separated by gel electrophoresis and blotted with antibodies to P70S6 kinase, p-P70S6 kinase (Thr389), or β-tubulin. Representative images of Western blot analysis are presented. <b>B.</b> The intensity of the bands was measured, and p-P70S6 protein levels were calculated relative to total P70S6 values showing a decrease in the level of P70S6 kinase phosphorylation. Data are the mean and SEM of 6 experiments. For serum containing groups the F and P values determined by one way ANOVA were 9.07 and 0.006 and for serum deprived groups were 10.18 and 0.005.</p

Neuroprotection of primary rat cortical neurons from a variety of insults.

<p>Primary rat cortical neurons were cultured in B27-supplemented neurobasal medium in the presence of 10 µM H₂O₂ (<b>A</b>), 0.5 µM colchicine (<b>B</b>), or 0.1 µM staurosporine (<b>C</b>) and different concentrations of human CysC for 24 hours. Cell survival analyzed by the MTS assay is expressed as percentage of live cells in cultures incubated in B27-supplemented medium without CysC. Data are the mean and SEM of 3 experiments. F and P values determined by one way ANOVA for (A) were 7.45 and 0.01. Differences between CysC treated and untreated samples were calculated by Student's t-test for B and C.</p

CysC forms lacking cathepsin B inhibitory activity are protective against nutrition-deprivation-induced cell death.

<p><b>A</b>. <i>In vitro</i> analysis of cathepsin B specific activity measured in the presence of 1.7 µM of three different forms of CysC: recombinant full length human CysC; recombinant full length CysC mutated in the inhibitory active site (mutated); and N-terminally truncated human urinary CysC (truncated). Cathepsin B inhibitory activity was calculated as activity per mg of total protein and normalized for cathepsin B protein level, presented as fluorescent units (fU). Data are the mean and SEM of 3 experiments. F and P values determined by one way ANOVA were 147.6 and <0.0001. <b>B</b>. Cortical primary neurons derived from embryos of CysC knockout mice were incubated in neurobasal medium either containing or lacking B27, in the presence of 0.8 µM of the three forms of CysC. Cell survival analyzed by the MTS assay is expressed as percentage of live cells in cultures incubated in supplemented media without CysC. Data are the mean and SEM of 3 experiments. F and P values determined by one way ANOVA were 9.39 and 0.005.</p