Cathepsin L Plays a Role in Quinolinic Acid-Induced NF-Κb Activation and Excitotoxicity in Rat Striatal Neurons

<div><p>The present study seeks to investigate the role of cathepsin L in glutamate receptor-induced transcription factor nuclear factor-kappa B (NF-κB) activation and excitotoxicity in rats striatal neurons. Stereotaxic administration of the N-methyl-d-aspartate (NMDA) receptor agonist Quinolinic acid (QA) into the unilateral striatum was used to produce the <i>in vivo</i> excitotoxic model. Co-administration of QA and the cathepsin L inhibitor Z-FF-FMK or 1-Naphthalenesulfonyl-IW-CHO (NaphthaCHO) was used to assess the contribution of cathepsin L to QA-induced striatal neuron death. Western blot analysis and cathepsin L activity assay were used to assess the changes in the levels of cathepsin L after QA treatment. Western blot analysis was used to assess the changes in the protein levels of inhibitor of NF-κB alpha isoform (IκB-α) and phospho-IκB alpha (p-IκBα) after QA treatment. Immunohistochemical analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced NF-κB. Western blot analysis was used to detect the effects of Z-FF-FMK or NaphthaCHO on QA-induced IκB-α phosphorylation and degradation, changes in the levels of IKKα, p-IKKα, TP53, caspase-3, beclin1, p62, and LC3II/LC3I. The results show that QA-induced loss of striatal neurons were strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced degradation of IκB-α, NF-κB nuclear translocation, up-regulation of NF-κB responsive gene TP53, and activation of caspase-3 was strongly inhibited by Z-FF-FMK or NaphthaCHO. QA-induced increases in beclin 1, LC3II/LC3I, and down-regulation of p62 were reduced by Z-FF-FMK or NaphthaCHO. These results suggest that cathepsin L is involved in glutamate receptor-induced NF-κB activation. Cathepsin L inhibitors have neuroprotective effects by inhibiting glutamate receptor-induced IκB-α degradation and NF-κB activation.</p></div

The effects of Z-FF-FMK and NaphthaCHO on QA-induced changes of IκB-α, p-IκB-α, IKKα and p-IKKα protein levels and cellular localization of NF-κB p65.

<p>Rats were pretreated with intrastriatal injection of Z-FF-FMK (5 nmol) or NaphthaCHO (5 nmol) 10 min prior to intrastriatal injection of QA (60 nmol) and were killed 12 h later. Control animals received vehicle injection only. A, B, C, D, E, F, G, H: Striatal tissues were dissected for preparation of striatal extracts for immunoblotting. Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and normalized with loading control (β-actin). Data are expressed as Mean ± SEM (n = 6). One-way ANOVA followed by Bonferroni <i>t</i>-test was used to carry out statistical comparisons. * <i>P</i><0.05 <i>vs.</i> control group; ** <i>P</i><0.01 <i>vs.</i> control group; # <i>P</i><0.05 <i>vs.</i> QA-treated group; ## <i>P</i><0.01 <i>vs.</i> QA-treated group. I: Brain sections were processed for immunofluorescence of NF-κB p65. A confocal microscope was used to examine brain sections. Note: NF-κB p65 expression was located in the cytoplasm in the control striatum (arrows). NF-κB p65 nuclear translocation was seen 12 h after QA administration (arrowheads). Pretreatment with the Cathepsin L inhibitor (Z-FF-FMK or NaphthaCHO) reduced QA-induced nuclear translocation of NF-κB p65. Scale bar  = 10 µm.</p

The effects of Z-FF-FMK and NaphthaCHO on QA-induced increases in P53 protein levels and activation of caspase-3.

<p>Rats were treated with intrastriatal injection of Z-FF-FMK (5 nmol) or NaphthaCHO (5 nmol) 10 min prior to QA (60 nmol) injection. Rats were killed 12 h later after QA injection. Striatal tissues were dissected for preparation of total lysates. The protein levels of P53 and active caspase-3 were determined with immunoblotting. Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i>-test. ** <i>P</i><0.01 <i>vs.</i> control group; ## <i>P</i><0.01 <i>vs.</i> QA-treated group.</p

The effects of Z-FF-FMK and NaphthaCHO on QA-induced increase in Beclin 1, LC3II/LC3I and decrease in P62 protein levels.

<p>Rats were treated with intrastriatal injection of Z-FF-FMK (5 nmol) or NaphthaCHO (5 nmol) 10 min prior to QA (60 nmol) injection. Rats were killed 12 h later after QA injection. Striatal tissues were dissected for preparation of total lysates. The protein levels of Beclin1, LC3II/LC3I and P62 were determined with immunoblotting. Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i>-test. * <i>P</i><0.05 <i>vs.</i> control group; ** <i>P</i><0.01 <i>vs.</i> control group; # <i>P</i> < 0.05 <i>vs.</i> QA-treated group; ## <i>P</i> < 0.01 <i>vs.</i> QA-treated group.</p

Activation of cathepsin L after QA treatment. A: The time-course of QA-induced increases in cathepsin L activity.

<p>Animals were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075702#pone-0075702-g003" target="_blank">Figure 3</a>. Cathepsin L activity in striatal lysates was determined with a fluorescence-based assay. <b>B: Effects of Z-FF-FMK and NaphthaCHO on cathepsin L activation.</b> Rats were pretreated with intrastriatal injection of Z-FF-FMK (5 nmol) or NaphthaCHO (5 nmol) 10 min prior to intrastriatal injection of QA (60 nmol) and were killed 12 h later. Control animals received vehicle injection only. Striata were dissected for assay of cathepsin L activity using a fluorescence-based assay kit. The results were expressed as percent of control (vehicle injection) after performing statistical analysis. Bars represent Mean ± SEM (N = 6). Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i>-test. * <i>P</i><0.05 vs. control group; ** <i>P</i><0.01 vs. control group; ## <i>P</i><0.01 vs. QA-treated group.</p

The effects of QA treatment on protein expression levels of cathepsin L, IκB-α and p-IκBα.

<p>Rats were treated with intrastriatal injection of QA (60 nmol) and killed 6, 12 and 24 h after drug administration. Striatal tissues were dissected for preparation of striatal extracts for immunoblotting. Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and normalized with loading control (β-actin). Data are expressed as Mean ± SEM (n = 6). Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i>-test. * <i>P</i><0.05 <i>vs.</i> control group; ** <i>P</i><0.01 <i>vs.</i> control group.</p

The effects of the cathepsin L inhibitor NaphthaCHO on QA-induced striatal damage.

<p>Rats were treated with intrastriatal injection of NaphthaCHO (2.5, 5, 10 nmol), 10 min prior to QA (60 nmol) injection. Rats were killed 14 days after QA treatment. Paraformaldehyde-fixed brain sections were stained with Nissl. <b>A:</b> The effects of NaphthaCHO on QA-induced striatal damage. Brain sections were photographed with a microscopy equipped with a CCD camera. Representative micrographs were taken in the center of drug injection (adjacent to needle tracks). <b>a and b:</b> Vehicle. <b>c and d:</b> QA. <b>e and f:</b> QA+ NaphthaCHO (2.5 nmol). <b>g and h:</b> QA+ NaphthaCHO (5 nmol). <b>i and j:</b> QA+ NaphthaCHO (10 nmol). b, d, f, h and j (×200) were enlarged from areas indicated with asterisks in a, c, e, g and i (×20). Scale bar  = 200 µm in (a, c, e, g and i);  = 20 µm in (b, d, f, h and j). <b>B:</b> Quantitative analysis of the effects of NaphthaCHO (2.5, 5, 10 nmol) on lesion size. Three Nissl-stained sections from each animal were used for quantitative analysis of lesion size caused by QA. Plate 1: sections taken at 1.4 mm anterior to the Bregma. Plate 2: sections taken at 0.6 mm anterior to the Bregma. Plate 3: sections taken at 0.2 mm posterior to the Bregma (The Stereotaxic Atlas of The Rat Brain by Xin-Min Bao, Si-Yun Shu; People's Health Press). Striatal images were captured and exported to Sigma Scan Pro 5 for determining lesion size. Lesion size was expressed as percent of the total size of the striatum of each section. Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i> test. <b>C:</b> The effects of NaphthaCHO on QA-induced loss of striatal neurons. 12 Nissl-stained sections (with the interval of every 14 successive brain sections) were used for counting neuronal numbers with an Optical Fractionator microscope and stereology software. The neuronal number of the total striatum was expressed as percent of control (vehicle-treated group). Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i> test. ** <i>P</i><0.01 <i>vs.</i> control group; # <i>P</i><0.05 <i>vs.</i> QA-treated group; ## <i>P</i><0.01 <i>vs.</i> QA-treated group.</p

The effects of the cathepsin L inhibitor Z-FF-FMK on QA-induced striatal damage.

<p>Rats were treated with intrastriatal injection of Z-FF-FMK (2.5, 5, 10 nmol), 10 min prior to QA (60 nmol) injection. Rats were killed 14 days after QA treatment. Paraformaldehyde-fixed brain sections were stained with Nissl. <b>A:</b> The effects of Z-FF-FMK on QA-induced striatal damage. Representative micrographs were taken in the center of drug injection (adjacent to needle tracks). <b>a and b:</b> Vehicle. <b>c and d:</b> QA. <b>e and f:</b> QA+Z-FF-FMK (2.5 nmol). <b>g and h:</b> QA+Z-FF-FMK (5 nmol). <b>i and j:</b> QA+Z-FF-FMK (10 nmol). b, d, f, h and j (×200) were enlarged from areas indicated with asterisks in a, c, e, g and i (×20). Scale bar  = 200 µm in a, c, e, g and i; scale bar  = 20 µm in b, d, f, h and j. <b>B:</b> Quantitative analysis of the effects of Z-FF-FMK (2.5, 5, 10 nmol) on lesion size. Three Nissl-stained sections from each animal were used for quantitative analysis of lesion size caused by QA. Plate 1: sections taken at 1.4 mm anterior to the Bregma. Plate 2: sections taken at 0.6 mm anterior to the Bregma. Plate 3: sections taken at 0.2 mm posterior to the Bregma (The Stereotaxic Atlas of The Rat Brain by Xin-Min Bao, Si-Yun Shu; People's Health Press). Striatal images were captured and exported to Sigma Scan Pro 5 for determining lesion size. Lesion size was expressed as percent of the total size of the striatum of each section. Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i> test. <b>C:</b> The effects of Z-FF-FMK on QA-induced loss of striatal neurons. 12 Nissl-stained sections (with the interval of every 14 successive brain sections) were used for counting neuronal numbers with an Optical Fractionator microscope and stereology software. The neuronal number of the total striatum was expressed as percent of control (vehicle-treated group). Bars represent mean ± SEM, n = 6 animals per group. Statistical comparisons were carried out with one-way ANOVA followed by Bonferroni <i>t</i> test. The difference was not statistically significant between QA and QA+Z-FF-FMK (2.5 nmol) treatment. * <i>P</i><0.05 <i>vs.</i> control group; ** <i>P</i><0.01 <i>vs.</i> control group; # <i>P</i><0.05 <i>vs.</i> QA-treated group; ## <i>P</i><0.01 <i>vs.</i> QA-treated group.</p

P188 improves long-term functional recovery after ischemia/reperfusion.

<p>(A) Post-stroke survival rate three weeks after stroke. *P<0.05, analyzed using log-rank test. (B) Wire hanging test. Time to stay hanging on the wire (T/hang). (C and D) Pole test. Time to turn the head downwards (T/turn). Time to reach the floor (T/floor). ^#, p<0.05 <i>vs.</i> sham-operated group; *, P<0.05 <i>vs.</i> saline-treated group. (E-G) Brain atrophy 3 weeks after tMCAO. Brain atrophy was determined using cresyl violet staining. The loss of brain volume was calculated as percentage of brain loss over total brain area of sham-operated mice.</p

P188 seals the membrane rupture of HT22 cell.

<p>Cells stained with PI indicated cells with disrupted membranes (PI⁺, A and D). Cells that remained permeable after P188 or PBS treatment showed green fluorescence (SYTOX® Green⁺, B and E ) or yellow in merged photograph (C and F ). Cell membrane resealed by P188 still showed red fluorescence in merged photograph but no green fluorescence (F).</p