Parthenolide and CNTF synergistically promote neurite growth of murine and primary adult human RGCs

A) Images of βIII-tubulin (tubulin) positive RGCs cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 0.5 nM or 5 nM). Scale bar: 15 μm

B) Quantification of neurite growth of cultures described in A with par concentrations between 0.25 and 5 nM. Data were normalized to untreated controls with an average neurite length of 4.6 μm per RGC and represent means ± SEM of six independent experiments.

C) Quantification of RGC numbers per well in cultures described in A.

D) Images of βIII-tubulin positive RGCs cultured for 4 days in the presence of vehicle (-) or par (0.5 nM), either without (con) or in combination with CNTF (200 ng/ml). Scale bar: 50 μm.

E) Quantification of neurite growth in cultures described in D. Data were normalized to untreated controls with an average neurite length of 9.6 μm per RGC and represent means ± SEM of seven independent experiments.

F) Representative images of βIII-tubulin (tubulin; green) positive RGCs and phosphorylated STAT3 (pSTAT3, red) in cultures described in D. Insets show higher magnifications of selected cells, indicated by white arrows. CNTF, but not par, induced STAT3 phosphorylation. Scale bars: 15 μm

G) Quantification of pSTAT3 positive RGCs in cultures described in D. Only CNTF, but not par, affected pSTAT3 levels. Data represent means ± SEM of three independent experiments.

H) Representative images of RGC neurites from cultures described in D, immunohistochemically stained for βIII-tubulin (green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm from axon tips after respective treatments. Scale bar: 15 μm.

I) Percentages of detyrosinated tubulin-positive (detyr+) axon tips. Par but not CNTF reduced detyrosination. Data represent means ± SEM of three independent experiments.

J) βIII-tubulin (tubulin) stained human RGCs isolated from the eyes of adult human patients and cultured for 4 days in the presence or absence of CNTF (200 ng/ml) and/or parthenolide (par, 0.5 nM). Scale bar: 100 µm.

K) Quantification of the average neurite length per RGC in cultures depicted in J. Par was applied at indicated concentrations, either alone or combined with CNTF. Data were normalized to untreated controls with an average neurite length of 16.3 µm per RGC and represent means ± SEM of four technical replicates, each from two independent experiments with individual human eyes (n=8).

L) Quantification of RGCs per well in cultures described in J, K.

M) Images of axon tips from RGCs as described in J. Cells were cultured in the absence (-) or presence of par (0.5 nM) and then immunostained for detyrosinated α-tubulin (detyr, red) and βIII-tubulin (tubulin, green). White arrows indicate detyrosinated tubulin-positive or negative neurite tips in control and the par-treated groups. Scale bar: 10 µm.

N) Quantification of axon tips containing detyrosinated tubulin from RGCs described in J. Thirty neurite tips per group were analyzed in three technical replicates from one-two independent experiments with individual human eyes (n=3-6).

Significances of intergroup differences in B, C, E, G, I, K, and N were analyzed using a one-way (B, C), two-way (E, G, I, K, N) analysis of variance (ANOVA) followed by the Tukey or Holm-Sidak post hoc test. P-values indicate statistical significance compared to the untreated (black p-values) or the CNTF-treated (red p-values) groups. n.s.=non-significant. Dots in B, C, E, G, and I represent values from at least three independent experiments. Dots in K, L, and N, represent values from technical replicates, each from two independent experiments.

hIL-6 elevates microtubule detyrosination

A) Schematic drawing illustrating the effect of hIL-6 on the PI3K/AKT/GSK3 signaling pathway. hIL-6 binding to gp130 activates phosphatidylinositol 3-kinase (PI3K), converting phosphatidylinositol (4, 5)-bisphosphate (PIP2) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). PIP3 stimulates AKT. Subsequent effects on GSK3, CRMP2, and microtubule detyrosination are shown in B-L.

B) Western blots of optic nerve lysates from untreated mice (con) or animals that had received intravitreal AAV2-hIL-6 injections 3 weeks earlier. Retinal hIL-6 expression elevated inhibitory phosphorylation of GSK3α and GSK3β, while total GSK3 levels remained unaffected. Inhibitory CRMP2 phosphorylation was reduced without altering total CRMP2 levels, while detyrosinated tubulin levels were increased. βIII-tubulin (tubulin) served as a loading control.

C-H) Densitometric quantification of western blots shown in B relative to βIII-tubulin and normalized to the untreated control (con). Data represent means ± SEM of samples from at least three animals per group.

I) Representative images of axon tips from dissociated RGCs from mice, as described in B. RGCs, were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of detyrosinated or negative axon tips after respective treatments. Scale bar: 15 μm

J) Quantification of the percentages of detyrosinated tubulin-positive (detyr+) axon tips. Par was applied at indicated concentrations. hIL-6 treatment increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments. P-values refer to untreated control.

K) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in

I, cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm

L) Quantification of the average neurite length per RGC in cultures depicted in K. Par was applied at indicated concentrations in combination with hIL-6. Data were normalized to untreated controls with an average neurite length of 8.4 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments.

Significances of intergroup differences were evaluated using Student’s t-test for quantifications shown in C-H. A one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test is shown in J and L, respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated hIL-6 (red p-values) groups. n.s.=non-significant. Dots represent values from single animals in C-H and three technical replicates of two independent experiments, respectively, in J and L.

PTEN−/− increases microtubule detyrosination

A) Schematic drawing illustrating the effect of PTEN−/− on the PI3K/AKT/GSK3 signaling path-way. PTEN−/− promotes the conversion of phosphatidylinositol (4, 5)-bisphosphate (PIP2) to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). PIP3 stimulates AKT and thus induces inhibitory GSK3 phosphorylation. As a result, the phosphorylation of CRMP2 and MAP1B is reduced. The effect on tubulin detyrosination is illustrated in B-E.

B) Representative images of axon tips from dissociated RGCs of PTEN-floxed mice. Animals were either untreated (con) or had received intravitreal injections of AAV2-Cre 3 weeks before dissociation (PTEN−/−). RGCs were cultured for 4 days in the presence of either vehicle (-) or parthenolide (par; 5 nM) and were immunohistochemically stained for βIII-tubulin (tubulin, green) and detyrosinated α-tubulin (detyr, red). White arrows indicate the last 15 μm of axon tips. Scale bar: 15 μm

C) Quantification of the percentages of detyrosinated tubulin-positive (detyr +) axon tips. Par was applied at indicated concentrations. PTEN−/− increased tubulin detyrosination, while par blocked this effect. Data represent means ± SEM of 3 technical replicates from 2 independent experiments.

D) Representative images of βIII-tubulin (tubulin) positive RGCs from cultures as described in B, cultured in the presence or absence of parthenolide (par; 0.5 nM or 5 nM). Scale bar: 50 μm

E) Quantification of the average neurite length per RGC in cultures depicted in D. Par was applied at indicated concentrations in combination with PTEN−/−. Data were normalized to untreated controls with an average neurite length of 1.04 µm per RGC and represent means ± SEM of three technical replicates from two independent experiments.

The significance of intergroup differences was evaluated through a one-way analysis of variance (ANOVA) followed by a Holm-Sidak or Tukey post hoc test for data shown in C and E, respectively. P-values indicate statistical significance compared to the untreated (black p-values) or the vehicle-treated PTEN−/− (red p-values) groups. n.s. = non-significant. Dots represent values from three technical replicates of two independent experiments.

DMAPT promotes optic nerve regeneration and enhances the effect of hIL-6

A) Timeline showing surgical interventions and intraperitoneal (i.p.) drug application for experiments presented in B-G.

B) Longitudinal optic nerve sections containing CTB (white) traced axons from mice 2 weeks after optic nerve crush (ONC) and daily repeated intraperitoneal vehicle (veh) or DMAPT injections, as described in A. Red asterisks indicate the lesion sites. Scale bar: 100 µm.

C) Quantification of regenerating axons at indicated distances distal to the lesion site in optic nerves as depicted in B, showing significantly enhanced optic nerve regeneration after DMAPT treatment compared to controls. Values represent the mean ± SEM of 6-7 animals per group (veh: n=6, dmapt: n=7).

D, E) Higher magnification of dashed boxes as indicated in B. Scale bar: 50 µm.

F) Confocal scans of retinal wholemounts from mice either uninjured (con) or subjected to ONC 14 days before tissue isolation. Animals received daily intraperitoneal injections of vehicle (veh) or DMAPT. RGCs were visualized by immunohistochemical βIII-tubulin staining (tubulin). Scale bar: 20 µm.

G) Quantification of RGC density in retinal wholemounts, as described in E. DMAPT, did not affect RGC survival in uninjured mice or after ONC. Values represent the mean ± SEM of 5-9 animals per group (veh, con: n=5; dmapt, con: n=5; veh ONC: n=7; dmapt, ONC: n=9).

H) Timeline showing surgical interventions and intraperitoneal (i.p.) drug application for experiments presented in I-K and Supplementary Fig. 3.

I) Immunostained retinal cross-sections prepared from mice that had received an intravitreal injection of AAV2-hIL-6-GFP simultaneously to optic nerve crush (ONC) and then daily intraperitoneal injections of vehicle or DMAPT (2 µg/kg) for 2 weeks. Sections were immunostained for phosphorylated STAT3 (pSTAT3, red). Transduction with AAV-2 hIL-6 is visualized by GFP (green) staining. hIL-6-induced STAT3-phosphorylation was not affected by DMAPT cotreatment. Scale bar: 20 µm.

J) Longitudinal optic nerve sections containing CTB (white) traced axons from mice described in A. Red asterisks indicate the lesion sites. Scale bar: 100 µm.

K) Quantification of regenerating axons distal to the lesion site at indicated distances in optic nerves from mice that received AAV2-hIL-6 combined with vehicle or DMAPT treatment, as shown in H and I. DMAPT significantly enhanced hIL-6-induced optic nerve regeneration. Values represent the mean ± SEM of 6-7 animals per group (veh: n=6; dmapt: n=7).

L-O) Higher magnification of dashed boxes as indicated in J. Scale bar: 50 µm.

Significances of intergroup differences in C, G, and K were evaluated using a two-way analysis of variance (ANOVA) with a Tukey post hoc test. P-values indicate statistical significance. Dots represent values from single animals.

DMAPT accelerates RpST regeneration and functional recovery after spinal cord crush

A) Timeline showing surgical interventions, BMS testing, and intraperitoneal (i.p.) drug application for experiments presented in B-K.

B) Representative photos showing open field hindlimb movement of mice at 1, 7, and 14 days post-injury (dpi). Before, mice had received spinal cord crush (SCC) and daily intraperitoneal injections of either DMAPT (dmapt) or the vehicle (veh).

C) BMS score of animals as described in A and B over 2 weeks after SCC. DMAPT significantly improved hindlimb movement’s functional recovery, resulting in plantar paw placement in most animals 2 weeks after spinal cord crush. Values represent the mean ± SEM of 13-17 animals per group (veh: n=13; dmapt n=17).

D) Immunohistochemical staining of transverse spinal cord sections from mice treated as described in A and B 2 weeks after injury. Serotonergic (5-HT) axons are present in the raphespinal tract proximal but not distal to the lesion site, indicating lesion completeness. Scale bar: 200 μm

E) Coronal thoracic spinal cord sections isolated from mice 2 weeks after SCC and daily intra-peritoneal injections of either dmapt or the veh. Raphespinal tract (RpST) axons were stained using an anti-serotonin antibody (5-HT, white). Only dmapt but not vehicle-treated mice showed regeneration of serotonergic axons > 2 mm beyond the lesion site (red arrow). Scale bar: 500 µm.

F-J) Higher magnifications of dashed boxes as indicated in E. Scale bar: 50 µm.

K) Quantification of regenerating 5-HT-positive axons from animals described in A and E at indicated distances beyond the lesion. Values represent the mean ± SEM of 7-8 mice per group (veh: n=8; dmapt: n=7).

Significances of intergroup differences in C and K were evaluated using a two-way analysis of variance (ANOVA) with a Tukey post-hoc-test. P-values indicate statistical significance.

Spinal cord regeneration and functional recovery after long-term DMAPT treatment

A) Timeline showing surgical interventions and intraperitoneal (i.p.) drug application for experiments presented in B-N.

B) Coronal thoracic spinal cord sections isolated from mice 8 weeks after spinal cord crush (SCC) and daily intraperitoneal injections of either DMAPT (dmapt) or the vehicle (veh). Raphespinal tract (RpST) axons were stained using an anti-serotonin antibody (5-HT, white). In contrast to the vehicle-treated control, dmapt enabled RpST regeneration over long distances beyond the lesion site (red arrow). Scale bar: 500 µm.

C-L) Higher magnification of dashed boxes indicated in B. Scale bar: 50 µm.

M) Quantification of regenerating 5-HT-positive axons as described in A and B at indicated distances beyond the lesion. Values represent the mean ± SEM of 7-10 animals per group (veh: n=7; DMAPT: n=10).

N) BMS score of animals as described in A and B, over 8 weeks after spinal cord injury. DMAPT significantly improved functional recovery of hindlimb movement, reaching a plateau 2 weeks after SCC. Values represent the mean ± SEM of 7-10 animals per group (veh: n=7; DMAPT: n=10).

Significances of intergroup differences in M and N were evaluated using a two-way analysis of variance (ANOVA) with a Tukey post-hoc-test. P-values indicate statistical significance.

DMAPT-mediated functional recovery is RpST-dependent

A) Validation of DHT-mediated depletion of serotonergic (5-HT, red) neurons in raphe nuclei (NRM = nucleus raphe magnus, NRPa=nucleus raphe pallidus, NRO=nucleus raphe obscurus) located above the pyramidal tracts in coronal medullary sections from mice as depicted in K, L 1 week after intracerebroventricular 5, 7-dihydroxytryptamine (DHT) injection, or untreated controls (con). Scale bar: 100 µm.

B-G) Higher magnification of indicated areas in images, as shown in A. Scale bar: 50 µm.

H) Quantification of serotonin (5-HT) positive raphe neurons per section in medullary immunostainings for untreated (con: n=5) and DHT-injected (DHT: n=8) mice as described in A.

I) Thoracic spinal cord cross-sections from animals, as described in A. Raphespinal axons were visualized by serotonin (5-HT) staining. Scale bar: 500 µm.

J) Quantification of 5-HT staining as sown in I for untreated (con: n=5) and DHT-injected (DHT, n=8) mice.

K) Representative images of vehicle-(veh) or DMAPT-(dmapt) treated mice at 7 or 21 d post-injury (dpi) and 1 day after DHT treatment.

L) BMS score of animals treated as described in (K) at indicated time points after spinal cord crush and DHT treatment. Values represent means ± SEM of 7-8 animals per group (veh: n = 8; dmapt: n=7), showing the average left and right hind paws score.

Significances of intergroup differences were evaluated using a two-way analysis of variance (ANOVA) with a Holm Sidak post hoc test (H, L) or Student’s t-test (J). The significance between group means is indicated by p-values. In L, comparisons are shown within the vehicle group (black), within the dmapt group (red), and between groups (blue).

DMAPT treatment accelerates hIL-6-induced RpST regeneration and functional recovery after spinal cord crush

A) Timeline showing surgical interventions and intraperitoneal (i.p.) drug application for experiments presented in B-K.

B) Representative photos showing open field hindlimb movement of mice at 1, 14, 35, and 56 days post-injury (dpi) of animals as described in A.

C) BMS scores of animals as described in A over 8 weeks after SCC. DMAPT treatment accelerated functional recovery of hindlimb movement in hIL6-treated mice. Values represent the mean ± SEM of 10 animals per group (veh: n = 10; DMAPT: n = 10).

D) Sagittal thoracic spinal cord sections isolated from mice described in A, 8 weeks after spinal cord crush (SCC), followed by intracortical AAV2-hIL-6 injection and daily intraperitoneal injection of either vehicle (veh) or DMAPT (dmapt). Raphespinal tract axons were stained using an anti-serotonin antibody (5-HT, white). DMAPT treatment did not significantly affect axon regeneration compared to vehicle-treated controls. Scale bar: 250 µm

E-J) Higher magnifications of dashed boxes as indicated in D. Scale bar: 100 µm

K) Quantification of regenerated 5-HT positive axons from animals described in A at indicated distances beyond the lesion site. DMAPT did not significantly enhance hIL-6-mediated raphespinal axon regeneration further. Values represent the mean ± SEM of 7-10 animals per group (veh: n = 7; DMAPT: n = 10).

Values represent means ± SEM of 7 or 10 (veh: n = 7; dmapt: n = 10) animals per group. Dots represent values for single animals. Significances of intergroup differences in C, K, and M were evaluated using a two-way analysis of variance (ANOVA) with a Tukey post-hoc-test. P-values indicate statistical significance.

Verification of human RGCs

A) Retinal flat mounts from human (left) and mouse (right) stained for βIII-tubulin. Scale bar: 50 μm.

B) Human (left) and mouse (right) RGCs cultured for 4 days and stained for βIII-tubulin. Scale bar: 25 μm.

C) Human βIII-tubulin-positive RGCs with regenerating neurites are also GAP43-positive. Scale bar: 50 μm

Parthenolide does not affect CRMP2 phosphorylation, STAT3 phosphorylation, or mTOR signaling

A) Representative photographs of dissociated RGCs stained for T514-phosphorylated CRMP2 (pCRMP2; red) and βIII-tubulin (tubulin; green) after 4 d in culture. RGCs were isolated from WT mice and either incubated with 0.5 nM parthenolide (par) or the vehicle (veh). Scale bar: 50 μm.

B) Quantification of pCRMP2 staining intensities of cultured RGCs as described in A. Integrated density of pCRMP2 staining was measured and normalized against values of the vehicle-treated control. Values represent means ± SEM of three independent experiments per group. In each experiment, 80 RGCs per group were analyzed in two replicate wells. Statistical analysis using Student’s t-test revealed no significant differences between the two groups.

C) Representative images of βIII-tubulin positive RGCs (green) and phosphorylated STAT3 (pSTAT3, red) from cultures as described in Fig. 2 I-L. hIL-6, but not par, induced STAT3 phosphorylation. Scale bar: 20 μm

D) Quantification of pSTAT3 positive RGCs in cultures described in C. Data represent means ± SEM of 3 technical replicates from 2 independent experiments.

E) Representative images of βIII-tubulin positive RGCs (tubulin; green) and phosphorylated S6 (pS6; red) from cultures as described in Fig. 3 B-E. PTEN−/−, but not par, induced S6 phosphorylation. Scale bar: 20 μm

F) Quantification of pS6 positive RGCs in cultures as described in C. Data represents means ± SEM of 3 technical replicates from 2 independent experiments.

Significances of intergroup differences in E and F were evaluated by a two-way analysis of variance (ANOVA) followed by a Tukey post hoc test. P-values indicate statistical significance. n.s.=non-significant. Dots represent values from three technical replicates of two independent experiments.

Optic nerve regeneration after AAV2-hIL-6 application and DMAPT treatment at suboptimal concentrations

A) Quantification of regenerating axons distal to the lesion site at indicated distances in optic nerves from mice that received AAV2-hIL-6 delivery combined with DMAPT treatment, as shown in 6 A. DMAPT was applied at 0.2 μg/kg. Regeneration was slightly enhanced compared to the vehicle-treated group (dashed line, as depicted in Fig. 4 C, D). Values represent the mean ± SEM of 4 animals.

B) Quantification of regenerating axons distal to the lesion site at indicated distances in optic nerves from mice that received AAV2-hIL-6 delivery combined with DMAPT treatment, as shown in 6 A. DMAPT was applied at 20 μg/kg. Regeneration was not improved compared to the vehicle-treated group (dashed line, as depicted in Fig. 4 C, D). Values represent the mean ± SEM of 5 animals.

Significances of intergroup differences in A and B were evaluated using a two-way analysis of variance (ANOVA) with a Tukey post hoc test. P-values indicate statistical significance. Dots represent values from single animals.

Verification of spinal cord lesion completeness

A) Schematic drawing showing the location of spinal cord cross sections proximal and distal to the T8 crush to identify potentially spared axons from the raphespinal tract (red).

B) Schematic drawing illustrating the expected pattern of serotonergic axons (red) on proximal spinal cord cross sections and an absence of axons on distal sections in case of a complete lesion.

C-H) Images of spinal cord cross sections proximal and distal to the crush site of animals as described in Fig. 5 A, Fig. 6 A, and Fig. 8 A. All animals in these experiments showed the expected pattern of serotonin-positive axons (5-HT, white) in the thoracic spinal cord > 3 mm proximal to the lesion site. No spared serotonergic axons were observed in the lumbar spinal cord > 8 mm distal to the lesion site, verifying the completeness of the crush injury. Scale bars: 500 µm.

DMAPT does not affect lesion size or CSPG release after SCC

A) Representative coronal spinal cord sections showing the T8 lesion site 8 weeks after SCC and daily intraperitoneal application of vehicle (veh) or DMAPT, as described in Fig. 6 A. Lesion sites and CSPGs were visualized by GFAP or CS-56 staining, respectively. Scale bar: 200 μm.

B-C) Quantification of the lesion area (B) and the lesion width (C) on spinal cord sections described in A. D) Quantification of the CS-56-positive (CS-56+) (CSPGs) area on spinal cord sections described in A. Statistical analyses in B-D were performed using a Student’s t-test. n.s. = non-significant. Data represent means±SEM of 6 (veh) or 5 (DMAPT) animals per group. Dots represent values for single animals.

DHT specifically depletes serotonergic neurons

A) Schematic of the Allen Brian Atlas illustrating the location of raphe nuclei (NRM= nucleus raphe magnus, NRPa = nucleus raphe pallidus) above the pyramidal tracts (Py) in a coronal view of the medulla. The white box indicates the location of images in B.

B) Sections from an untreated animal (con) and a mouse 1 day after intracerebroventricular 5,7-dihydroxytryptamine (DHT) injection. Neurons are immunostained for neuN (green), while Raphe neurons are visualized by serotonin (5-HT, red) immunostaining. Dapi (blue) labels all nuclei. DHT treatment depletes serotonergic neurons with only very few 5-HT positive neurons remaining (white arrows), while other neuN positive neurons are unaffected. Scale bar: 250 µm.

DMAPT does not affect hIL-6-mediated STAT3 activation

A) Schematic of a coronal brain section with the AAV2-hIL-6 injection sites (dashed box) shown in B.

B) Section of layer V sensorimotor cortex isolated from mice 8 weeks after spinal cord crush (SCC), intracortical AAV2-hIL-6 injection, and subsequent daily intraperitoneal injections of either vehicle (veh) or DMAPT (dmapt). GFP (green) was co-expressed by AAV2-hIL-6. The tissue was immunohistochemically stained for phosphorylated STAT3 (pSTAT3, red). DMAPT treatment did not affect STAT3 phosphorylation. Scale bar: 50 μm

C-H) Higher magnification of dashed white boxes indicated in B. Scalebar: 50 µm.

I) Schematic drawing of a coronal medulla section showing transneuronal stimulation (STAT3 phosphorylation) of neurons in raphe nuclei (RN) with hIL-6 secreted from collateral sprouts from the pyramidal tract (PY).

J) Section of the medulla showing mice’s medullary raphe nuclei (RN) as described in B. The tissue was immunohistochemically stained for pSTAT3 (red) and 5-HT (green). STAT3 phosphorylation indicates transneuronal stimulation of raphe neurons with hIL-6 secreted from pyramidal axon sprouts (PY). DMAPT treatment did not affect pSTAT3 levels. Scale bar: 50 µm. K-P) Higher magnification of dashed white boxes indicated in J. Scalebar: 100 µm.