Treatment of MOG_35–55 induced EAE by CpdA.

<p>(A) EAE was induced in C57Bl/6 mice followed by treatment with 15 mg/kg CpdA (dissolved in 20% ethanol), 100 mg/kg Dex or PBS as a control (con) on 3 consecutive days starting at an average clinical score of 2 (marked by arrows); n = 11; statistical analysis: days 11–22. The cross indicates that all animals of this group either died or had to be sacrificed for ethical reasons. The experiment was repeated twice with similar results. (B) CpdA was dissolved in water and therapeutically applied at doses of 5 mg/kg or 1.5 mg/kg for 3 consecutive days after the mice had developed an average clinical score of 3 (marked by arrows); treatment with the solvent alone (con) served as a control; n = 18/6/19; statistical analysis: days 13–23. The experiment was repeated five times with similar results. (C) EAE was induced in C57Bl/6 and GR^lckCre mice followed by treatment with CpdA dissolved in water at a dose of 5 mg/kg or the solvent alone (con). Therapy was started at an average score of 2 (defined as day 0); n = 6; statistical analysis: days 1–10. The experiment was repeated twice with similar results. (D) C57Bl/6 wildtype or GR^lckCre mice were treated with 5 mg/kg or 15 mg/kg CpdA dissolved in water or PBS as a control on 3 consecutive days. On the following morning surviving mice were sacrificed and the cellularity of the spleen was determined by microscopic counting. n = 3/12. **: p<0.01, ***: p<0.001, n.s.: p>0.05.</p

CpdA-induced apoptosis is independent of the GR.

<p>(A,B) Thymocytes were isolated either from GRN^+/+ or GRN^−/− E18.5 embryos and cultured in the absence (con) or presence of different concentrations of Dex (A) or CpdA (B) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3. (C,D) WEHI 7.1 cells stably transduced with a retrovirus encoding a GR-specific shRNA (GR-siRNA), Bcl-2 or the empty retroviral vector LMP, were cultured in the absence (con) or presence of different Dex concentrations (C) or 10⁻⁵ M CpdA (D) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3−5. *: p<0.05, **: p<0.01, n.s.: p>0.05.</p

Mechanism of CpdA action in the treatment of EAE.

<p>(A) EAE was induced in C57Bl/6 mice followed by treatment with 1.5 mg/kg or 5 mg/kg CpdA dissolved in water for 3 consecutive days after the mice had developed an average clinical score of 3. On the following morning the mice were sacrificed and the leukocytes isolated from the spinal cord. Cellularity was determined by microscopic counting; staining for AnnexinV binding and surface expression of LFA-1 on CD3⁺CD4⁺ Th cells was performed by flow cytometry. n = 3−7. (B) The spleen was isolated from the same animals as in panel A and the leukocytes analyzed for AnnexinV binding as well as LFA-1 and CD44 surface expression on CD3⁺CD4⁺ Th cells by flow cytometry. n = 3−8. (C, D) Splenocytes from the same animals as in panels A and B were cultured in the presence or absence of ConA or MOG_35–55 peptide. Proliferation was measured by ³[H]-thymidine incorporation assay and expressed as a proliferation index relative to the values obtained in the absence of any stimulus (C); IL-17 and IFNγ levels in the supernatant were determined by ELISA (D); n = 7−10. *: p<0.05, ***: p<0.001, n.s.: p>0.05.</p

CpdA exerts pro-apoptotic activity in various cell-types.

<p>(A) Thymocytes were cultured in the presence or absence of 10⁻⁶ M Dex or 10⁻⁵ M CpdA for 24 hrs. Apoptosis was assessed at different time points by flow cytometry based on staining for AnnexinV/7-AAD. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3. (B) Thymocytes were treated with Dex or CpdA for 24 hrs as in panel A, either in the absence (con) or the presence of 100 µM Z-VAD-fmk (pan-caspase inhibitor). n = 3. (C) SK-N-SH neuroblastoma cells were treated with Dex or CpdA at concentrations of 10⁻⁶ M and 10⁻⁵ M for 24 hrs or left untreated (con). To test whether CpdA action depends on caspase-activity, the experiment was additionally performed in the presence of 100 µM Z-VAD-fmk. n = 6. (D) 5×10⁴ MEFs generated from GRN^+/+ and GRN^−/− fetuses were cultured in the absence (con) or presence of 10⁻⁶ M Dex or 10⁻⁵ M CpdA. After 48 hrs cell numbers were determined by microscopic counting. n = 3. *: p<0.05, **: p<0.01, ***: p<0.001, n.s.: p>0.05.</p

Inhibition of microglial cytokines and chemokines production by MB.

<p>Primary cultures of control (newborn control: ctrl_p0; adult control from corresponding siblings of mutant mice: ctrl_p120) or mutant (from advanced clinical SOD1^G93A mice; G93A_p120) microglia (15,000 cells/well) were incubated with MB (1, 10, 20, 40 and 100 µM) in the presence or absence of LPS (100 ng/ml) for 18 hours. Cytokine and chemokine release profile was determined in the supernatants. Data are presented relative to the corresponding value of LPS-triggered cytokine or chemokine release from control microglia of newborn mice (set as 1). Data are mean ± SEM of triplicates of 3 (MCP-1), 4 (IL-6) or 5 (TNF-α, RANTES, KC, MIP-1α and IL-12) independent experiments; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001).</p

Delayed onset of disease by oral application of MB.

<p>(A) Control and MB-treated (3, 10, 30 and 100 mg oral per kg body weight per day) SOD1^G93A mice with respect to the different stages of disease (stage 1: onset of disease/early clinical stage; stage 2: clinical stage; stage 3: advanced clinical stage). (B and C) Weight and survival profiles for SOD1^G93A mice (control and MB-treated). In B the data are presented relative to the highest value in each group. Values are presented as mean ± SEM; ctrl: n = 16 mice, 3 mg: n = 12, 10 mg: n = 10, 30 mg: n = 8, 100 mg: n = 11; ANOVA followed by Tukey test (*p<0.05, **p<0.01).</p

Effect of systemic application of MB on intracellular TDP-43-containing aggregates.

<p>To analyze intracellular aggregations in anterior horn neurons, TDP-43 and SMI-32 staining was performed in lumbar cross-sections. TDP-43-containing aggregates in SMI-32-positive neurons were observed in the anterior horn of control and MB-treated pre-clinical and advanced clinical mice. An exemplary TDP-43-stained neuron from each group, marked by an arrow, is shown in the last line of images.</p

Delayed motor disturbance by MB application.

<p>Rotarod motor performance of untreated and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1^G93A mice. The time until mice fell off the rotarod at 12 rpm (A) and the velocity the mice reached using an acceleration rate of 1 rpm every 10 s (B) are presented. Each animal was tested three times per trial. Values are presented as mean ± SEM; both groups: n = 10 mice; ANOVA followed by Tukey test (*p<0.05, **p<0.01).</p

Delayed onset of disease by intraperitoneal application of MB.

<p>(A) Control and MB-treated (10 mg intraperitoneal injection per kg body weight per day) SOD1^G93A mice with respect to the different stages of disease. (B and C) Weight and survival profiles for SOD1^G93A mice (control and MB-treated). In B the data are presented relative to the highest value in each group. Values are presented as mean ± SEM; both groups: n = 16 mice; ANOVA followed by Tukey test (*p<0.05, ***p<0.001).</p

Effect of systemic application of MB on motor neuron survival.

<p>(A) NeuN-stained cross-sections of the spinal cord at the level L3 to L5. Non-treated controls (SOD1^G93A mice) are depicted left. Corresponding sections of MB-treated SOD1^G93A mice are shown on the right (10 mg oral per kg body weight per day; drug administration started at the age of 45 days) (B) Counting of neurons in the anterior horn of SOD1^G93A mice at different disease stages. Cell somata bigger than 20 µm were counted and given per mm². In preclinical stages, neuron number was significantly higher in MB-treated mice compared to non-treated mice indicating an early neuroprotective effect of MB. Note that no differences were observed in later disease stages. Values are presented as mean ± SEM; n = 4 mice for each group; ANOVA followed by Tukey test (*p<0.05).</p