Article Figures & Data
Figures
- Figure 1.
URMC-099 prevents excitatory synapse loss in EAE hippocampus. A, Staining for PSD95-positive postsynaptic puncta in the stratum radiatum of hippocampal area CA1 (inset) was decreased in vehicle-treated EAE mice relative to sham-immunized controls. Treatment with URMC-099 beginning after the development of EAE motor deficits preserved synaptic puncta, while treatment with CLFB1134 did not. Scale bar: 10 µm. B, Density of PSD95-positive puncta, quantified relative to sham-immunized controls; *p < 0.05, one-way ANOVA with Sidak post hoc test. C, Treatment with 099 and 1134 in this cohort resulted in a modest reduction in mean severity of EAE motor deficits that was not significantly different from controls; n = 10 male mice (sham-vehicle), n = 6 male mice (EAE-vehicle and EAE-099), n = 7 male mice (EAE-1134). Data in all figures are expressed as mean ± SEM.
- Figure 2.
URMC-099 modulates the phenotype of activated microglia in EAE hippocampus. A, Staining for Iba1 in hippocampal area CA1 shows ramified microglia in sham-immunized mice, and microglia with shorter, thicker processes in EAE. B, Iba1-positive cells in the hippocampi of both sham and EAE mice coexpress microglia-specific marker Tmem119 along their processes and to a more variable extent in the cell body (upper panels). Iba1-positive, Tmem119-negative cells consistent with monocyte-derived macrophages could be found in the pia during EAE (inset), but not in or around the hippocampus. Staining for Ly-6B, expressed on neutrophils and some recently-generated macrophages, is sparse in sham and EAE hippocampi (lower panels, arrowheads), although clusters occurred sporadically in the pia and other area of some EAE brains (inset). Scale bars: 20 µm. s.o., CA1 stratum oriens; pyr, pyramidal layer; s.r., stratum radiatum. C, Quantification of microglial images shows increased Iba1 intensity and reduction in the ratio of surface area to volume in EAE microglia regardless of treatment with URMC-099 or CLFB1134. Microglial expression of lysosomal marker CD68 is increased in many hippocampi from vehicle-treated EAE mice but not in mice treated with URMC-099 or CLFB1134; n = 10 male mice (sham-vehicle), n = 6 male mice (EAE-vehicle and EAE-099), n = 7 male mice (EAE-1134). D, E, Western blottings and band densitometry for markers of inflammation and microglial phenotype in hippocampal protein extracts. Markers associated with proinflammatory microglial activation tended to increase in vehicle-treated EAE hippocampi and were decreased by URMC-099, with significant changes in iNOS (inducible nitric oxide synthase) and immunoglobulin receptor FCγR1/CD64 (lower single band), and a non-significant trend toward increased pro-IL1β. Proinflammatory marker CD86 does not significantly increase in vehicle-treated EAE but is decreased by URMC-099 treatment. Arg1 and IL-10, canonical markers of anti-inflammatory alternative activation, are not significantly changed in EAE or with URMC-099. Consistent with immunostaining results, Iba1 is up-regulated in vehicle-treated EAE hippocampi and is further increased with URMC-099 treatment. Complement component C1q follows a similar pattern; n = 10 male mice (sham-vehicle), n = 7 male mice (EAE-vehicle), n = 11 male mice (EAE-099); one representative blot with three lanes per condition is depicted; *p < 0.05, **p < 0.01, one-way ANOVA with Sidak (iNOS, FCγR1/CD64, CD86) or Tukey (Iba1, SA/Vol, C1q) post hoc test.
- Figure 3.
URMC-099 protects neuronal cultures during growth factor deprivation. A, Phase-contrast images (upper) of cultured SCG neurons show disruption of neurite integrity after 48 h of NGF deprivation, and Hoechst DNA staining (lower) shows condensed pyknotic nuclei (arrowheads indicate examples) in the same neurons. Cultures treated with URMC-099 (300 nM) maintained neurite integrity and diffuse nuclear chromatin staining indicative of healthy neurons after 48 h of NGF deprivation. Cultures treated with CLFB1134 had marked fragmentation of neurites, and cellular damage severe enough to preclude Hoechst analysis. Scale bars: 60 µm. B, Quantification of dead SCGs identified by pyknotic nuclei shows a dose-dependent protective effect for URMC-099 following NGF deprivation (n = 4 cultures for each condition). C, Western blottings for JUN, which mediates SCG apoptosis during NGF withdrawal and is activated downstream of MLK signaling via phosphorylation by JNK, show increased JUN expression and phosphorylation at serine residues 63 and 73 in protein isolates from SCG cultures following NGF withdrawal, which is nearly abolished by treatment with URMC-099 (300 nM, n = 3 experiments); *p < 0.05, **p < 0.0001, one-way ANOVA with Sidak post hoc test.
- Figure 4.
URMC-099 preserves contextual fear conditioning in EAE mice. A. Contextual and cued fear conditioning protocol for EAE mice. Mice were conditioned in an isolation chamber where they were presented three times with an auditory cue paired with a foot shock; 24 h later, mice were evaluated for episodes of freezing after being returned to the identical chamber (conditioned context), introduced to a chamber modified in shape, texture and odor (novel context), and presented with the auditory cue (cue). B, Group averages of freezing responses during each of the three tests, expressed as % of time spent freezing in 30-s epochs. Mean rates of freezing (dotted lines) increased in the conditioned context and with cue presentation relative to the unconditioned novel context in all groups, although EAE mice with motor deficits had higher rates of immobility in the novel context compared to sham-immunized controls. Mean freezing rates in the novel context were subtracted from those in the conditioned context and during cue presentation (Δ freeze) to differentiate freezing due to conditioning from non-specific immobility. C, Δ freeze values show that deficits in contextual fear conditioning in vehicle-treated EAE mice were restored to control levels by URMC-099 treatment. EAE mice showed no deficits in auditory cue conditioning, with similar Δ freeze values in all groups; *p < 0.05, two-way ANOVA with Sidak post hoc test of the treatment main effect. D, EAE scores for the mouse cohorts that underwent behavioral testing show that URMC-099 treatment after symptom onset did not affect peak EAE severity and was associated with modest recovery that was statistically different from vehicle-treated mice after 9–13 d of disease; *p < 0.5, Mann–Whitney U test. For all panels, n = 29 mice (sham-vehicle), n = 18 mice (EAE-vehicle), n = 21 mice (EAE-099).
Tables
Comparison Data structure Type of test Mean diff. (95% CI) a PSD95, EAE-V versus Sham-V Normal One-way ANOVA, Sidak post hoc –0.24 (–0.45 to –0.02) b PSD95, EAE-099 versus EAE-V Normal One-way ANOVA, Sidak post hoc 0.25 (0.01 to 0.48) c EAE score (IHC cohort), EAE-099 versus EAE-V Non-parametric Mann–Whitney U test –0.5 (–2.5 to 0.5)* d EAE score (conditioning cohort), EAE-099 versus EAE-V Non-parametric Mann–Whitney U test –1 (–1 to 0)* e Microglia SA:volume, EAE-V versus Sham-V Normal One-way ANOVA, Tukey post hoc –0.98 (–1.54 to –0.43) f Microglia SA:volume, EAE-099 versus Sham-V Normal One-way ANOVA, Tukey post hoc –1.09 (–1.64 to –0.53) g Microglia SA:volume, EAE-1134 versus Sham-V Normal One-way ANOVA, Tukey post hoc –0.81 (–1.33 to –0.28) h Iba1, EAE-099 versus Sham-V Normal One-way ANOVA, Tukey post hoc 0.44 (0.05 to 0.82) i CD68, EAE-V versus Sham-V Normal One-way ANOVA, Tukey post hoc 0.73 (–0.26 to 1.71) j iNOS, EAE-V versus Sham-V Normal One-way ANOVA, Sidak post hoc 0.93 (0.29 to 1.57) k iNOS, EAE-099 versus EAE-V Normal One-way ANOVA, Sidak post hoc –0.61 (–1.23 to –0.01) l FcyR1-CD64, EAE-V versus Sham-V Normal One-way ANOVA, Sidak post hoc 0.56 (0.16 to 0.95) m Pro-IL1β, EAE-V versus Sham-V Normal One-way ANOVA, Sidak post hoc 0.87 (–0.02 to 1.77) n CD86, EAE-099 versus EAE-V Normal One-way ANOVA, Sidak post hoc –0.22 (–0.39 to –0.05) o Iba1, EAE-V versus Sham-V Normal One-way ANOVA, Tukey post hoc 5.69 (0.63 to 10.75) p Iba1, EAE-099 versus EAE-V Normal One-way ANOVA, Tukey post hoc 7.75 (3.27 to 12.24) q C1q, EAE-V versus Sham-V Normal One-way ANOVA, Tukey post hoc 2.63 (0.62 to 4.63) r C1q, EAE-099 versus Sham-V Normal One-way ANOVA, Tukey post hoc 3.01 (1.37 to 4.65) s Neuronal apoptosis, 100 nM 099 versus vehicle (24 h) Normal One-way ANOVA, Sidak post hoc –37.0 (–23.2 to –50.8) t Neuronal apoptosis, 300 nM 099 versus vehicle (24 h) Normal One-way ANOVA, Sidak post hoc –38.2 (–24.4 to –52.0) u Neuronal apoptosis, 100 nM 099 versus vehicle (48 h) Normal One-way ANOVA, Sidak post hoc –40.8 (–27.0 to –54.6) v Neuronal apoptosis, 300 nM 099 versus vehicle (248 h) Normal One-way ANOVA, Sidak post hoc –53.9 (–40.1 to –67.7) w P Ser 73 JUN, 099 versus vehicle (8h) Normal One-way ANOVA, Sidak post hoc –0.80 (–0.14 to –1.45) x P Ser 73 JUN, 099 versus vehicle (12h) Normal One-way ANOVA, Sidak post hoc –0.99 (–0.33 to –1.64) y Context Δ freeze, EAE-V versus Sham-V (treatment main effect) Normal Two-way ANOVA, Sidak post hoc –0.33 (–0.61 to –0.05) z Context Δ freeze, EAE-099 versus EAE-V
(treatment main effect)Normal Two-way ANOVA, Sidak post hoc 0.32 (0.02 to 0.62) ↵* Mann–Whitney U test results reported for days with greatest mean difference between group scores (IHC cohort: days 5–6; fear conditioning cohort: days 10–12).
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