Thiorphan reprograms neurons to promote functional recovery after spinal cord injury

Abstract

We previously identified an embryonic shift in the corticospinal motor neuronal transcriptome after spinal cord injury associated with successful axonal regeneration¹. Exploiting this transcriptional regenerative 'signature', here we used in silico screens to identify small molecules that generate similar shifts in the transcriptome, and identified thiorphan-a neutral endopeptidase inhibitor-as a lead candidate. In a new adult motor cortex neuronal in vitro screen², thiorphan increased neurite outgrowth 1.8-fold (P < 0.001). We then infused thiorphan into the central nervous system beginning 2 weeks after severe C5 spinal cord contusions and, when combined with a neural stem cell graft, thiorphan elicited significant improvements in forelimb function (P < 0.005) and corticospinal regeneration (P < 0.05). Extending clinical relevance, thiorphan significantly increased neurite outgrowth in primary cortical neuronal cultures from a 56-year-old human. These findings represent a new path for drug discovery, starting from in silico screens to proof-of-concept in adult human brain cultures.

Fig. 1. Drug discovery pipeline, in silico analysis and in vitro validation.

a, Drug discovery pipeline consisting of five steps: (1) creation of a transcriptomic dataset; (2) in silico analysis using CMap; (3) medium-throughput in vitro screen; (4) in vivo testing of lead candidate(s); and (5) in vitro monkey and human validation. CST, corticospinal tract. b, In silico analysis shows ranking of compounds in CMap on the basis of connectivity score (left y axis), specificity score (blue, right y axis) and reliability score (green, right y axis) (see text for further detail). c, Neurite extension in composite image of cortical neurons treated with DMSO control and thiorphan (250 μM), Tuj1 labelling. d,e, In vitro screen of top three ‘hits’ from in silico screen, together with predicted negative modulator, adiphenine. Tested in cultures of adult mouse primary cortical neurons for 5 days in vitro, three independent biological replicates were performed on separate days, each using neurons from four adult mice. For each condition, data from all neurons measured across replicates were combined to yield n = 200 neurons per condition. Each neuron was spatially separated and analysed as an independent observation. Total neurite outgrowth per cell (Tuj1 labelling) is shown for each condition. Median values: DMSO (82.9); thiorphan (90.7, 99.8, 132.4, 166.1); triflusal (74.3, 61.7, 94.5, 124.5); milrinone (78.9, 77.4, 68.5, 75.5); adiphenine (74.4, 44.5, 31.7, 3) (d). Maximum neurite length per cell is shown for each condition. Median values: DMSO (93.3); thiorphan (111.1, 105.4, 122.1, 128); triflusal (84.4, 85, 104.9, 114.3); milrinone (100.6, 81.7, 79, 78.6); adiphenine (87.4, 60, 30.6, 6.23) (e). Statistical significance was determined by two-tailed Student’s t-test (**P < 0.01, ***P < 0.001). Error bars ± s.e.m. (d,e). Scale bar, 25 μm (c).

Fig. 2. Thiorphan improves functional outcomes after severe C5 bilateral contusion.

a, In vivo experimental outline of a clinically relevant SCI model. b, Nissl stain of a severe C5 bilateral contusion model. Sagittal section, rostral to left. Top, large contusion cavity in an ungrafted animal after 3 months. Bottom, NPC graft filling the lesion cavity and providing substrate for potential corticospinal axon regeneration. c, Skilled forelimb successful pellet retrieval with right paw on Montoya staircase ± s.e.m. Animals that received thiorphan and a substrate for axonal regeneration into the lesion site—an NPC graft—exhibit significant functional recovery over time compared with lesioned controls (P = 0.005, group × time interaction, Poisson generalized linear model). Treatment with thiorphan alone also trended towards significance (P = 0.14). The lesion model applied in this experiment is the most severe that we have tested, and NPC grafts also exhibited a trend towards better outcomes than lesioned controls but this did not reach statistical significance. Grey shading, period of thiorphan infusion into cortex. d, Thiorphan + NPC graft group also exhibits significant recovery of pellet retrieval accuracy in this severe lesion model (P = 0.001). Other treated groups trend towards improved outcomes compared with lesioned controls. Accuracy represents the number of pellets eaten divided by the number of pellets displaced plus the number of pellets eaten. *P < 0.05 (c,d). Scale bar, 500 μm. Source data

Fig. 3. Thiorphan increases corticospinal axonal regeneration.

a, CST axons labelled anterogradely with Flag (red) show greater regeneration into E14 neural progenitor stem cell grafts (green (green fluorescent protein (GFP))) in animals with thiorphan cortical infusions. The white dotted line indicates the host–graft interface; the boxed region is shown at higher magnification in c. b, Fewer corticospinal axons regenerate into animals that did not receive thiorphan cortical infusions. The white dotted line indicates the host–graft interface; the boxed region is shown at higher magnification in d. c,d, Higher magnification views of the boxed regions in a (c) and b (d) showing regenerating corticospinal axons. e, Corticospinal regeneration into graft occupying lesion site is increased 1.6-fold in the presence of thiorphan infusion into the motor cortex (*P < 0.05, two-tailed t-test). In the absence of a graft, no host axons are present in the lesion site; non-grafted animals could not be quantified. f, Regenerating host corticospinal axons form putative synapses with grafted neurons, based on colocalization of regenerating CST (red) with synaptophysin (SYN, white) apposed to grafted neuron (GFP, green). DAPI in blue. Arrows indicate the exact orthogonal view intersection point, single plane. Scale bars, 500 μm (a,b); 200 μm (c,d); 5 μm (f). Source data

Fig. 4. Thiorphan increases growth of monkey and human cortical neurons and induces pro-regenerative state.

a, Thiorphan significantly increased total neurite outgrowth from primary cultures of adult cynomolgus macaque motor cortex labelled for Tuj (***P < 0.001, two-tailed t-test). Control cultures received DMSO. Each point represents the total neurite length of individual neurons. Samples were run as technical duplicates, with neurons cultured for 5 days. b, Thiorphan treatment also significantly increased the maximum neurite length per cell by 1.8-fold (****P < 0.0001, two-tailed t-test). c, Representative images of cultures of adult primate motor cortex treated with diluent or thiorphan. d, Thiorphan significantly increased total neurite outgrowth from primary cultures of normal 56-year-old human cortex neurons labelled for Tuj (****P < 0.0001, two-tailed t-test). Neurons cultured for 5 days. Each point represents the total neurite length of one neuron. e, Thiorphan treatment of human cortical neurons also significantly increased the maximum neurite length per cell (**P < 0.01, two-tailed t-test). f, Representative images of 56-year-old human cortical neurons in culture labelled for Tuj1. g, RNA sequencing was performed on cultures of adult primary motor cortex from rhesus monkeys treated for 5 days with thiorphan or vehicle (DMSO). A total of 177 genes changed ±1.5-fold on a log₂ scale. h, Gene ontology of these 177 genes demonstrates clear enrichment for developmental processes (Gene Ontology database, 10.5281/zenodo.10536401). i, To identify in vivo mechanisms recruited by thiorphan treatment, rat brains were removed after 2 weeks of thiorphan infusion and compared with diluent-infused controls. Thiorphan treatment increased immunolabelling for both BDNF and phospho-AKT in the infused motor cortex, reflecting a shift to a state that is developmental and pro-regenerative. Scale bars, 20 μm (c,f). Scale bars, 25 μm, inset scale bars, 10 μm.