Tppp3 is a novel molecule for retinal ganglion cell identification and optic nerve regeneration

Abstract

Mammalian central nervous system (CNS) axons cannot spontaneously regenerate after injury, creating an unmet need to identify molecular regulators to promote axon regeneration and reduce the lasting impact of CNS injuries. While tubulin polymerization promoting protein family member 3 (Tppp3) is known to promote axon outgrowth in amphibians, its role in mammalian axon regeneration remains unknown. Here we investigated Tppp3 in retinal ganglion cells (RGCs) neuroprotection and axonal regeneration using an optic nerve crush (ONC) model in the rodent. Single-cell RNA sequencing identified the expression of Tppp3 in RGCs of mice, macaques, and humans. Tppp3 overexpression enhanced neurite outgrowth in mouse primary RGCs in vitro, promoted axon regeneration, and improved RGC survival after ONC. Bulk RNA sequencing indicated that Tppp3 overexpression upregulates axon regeneration genes such as Bmp4 and neuroinflammatory pathways. Our findings advance regenerative medicine by developing a new therapeutic strategy for RGC neuroprotection and axon regeneration.

Keywords: Axon regeneration; BMP4; Inflammation; Neurite outgrowth; Retinal ganglion cells; Tppp3.

Fig. 1

Identification of Tppp3 by single-cell RNA sequencing. A Schematic representation of the Experimental design for single-cell RNA sequencing (scRNA-seq) conducted on E14.5 retina samples treated with GDF11 or PBS. B t t-distributed stochastic neighbor embedding (t-SNE) visualization of retinal progenitor cells, with cells color-coded based on their cluster assignments and treatment conditions. Cluster 3 represents the RGC cluster. C GDF11, RGC fate suppressor protein, leads to a reduction in the expression levels of Pou4f1 and Tppp3, specifically within the RGC cluster (cluster 3). This highlights the potential role of Tppp3 in RGC differentiation. D Pou4f1 expression is specifically localized within cluster 3, identified as the RGC-specific cluster. E Tppp3 is also highly expressed within cluster 3. F Violin plot displaying the expression level of Tppp3 from a reanalysis of scRNA data obtained from purified RGCs. The X-axis represents the time points following optic nerve crush, while the numbers above the violin plots indicate the percentage of RGCs expressing Tppp3. 2 weeks after ONC, Tppp3 expression is reduced substantially. Tppp3 is highly expressed within the RGC clusters of G macaque and H humans

Fig. 2

TPPP3 is expressed in mouse RGCs and promotes RGC neurite outgrowth ex vivo. A Western blots show that RGC markers BRN3A, RBPMS, and THY1 are expressed selectively in the immunopurified RGC cell population, as is Tppp3. B Immunostaining of RGCs for BRN3A and TPPP3 in adult mouse retinal sections reveals that Tppp3 is expressed within the RGC layer. ~ 75% of BRN3A+ cells co-express Tppp3 (white arrows). Scale bar = 100 µm. C Immunostaining of P2 primary RGCs shows expression of anti-β-III-tubulin antibody E7. Co-labeling with anti-β-III-tubulin antibody E7 and Tppp3 confirmed that Tppp3 is expressed primarily within the soma of RGCs. Scale bar = 50 µm. D RNAscope analysis of Tppp3 in the developing mouse eye. Tppp3 expression reached its peak at E14.5 and subsequently decreased. E Representative images of primary RGCs transduced with AAV2-control or AAV2-Tppp3-OE vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 overexpression increases RGC neurite outgrowth by ~ 20% (n = 5 independent cultures). F Representative images of primary RGCs transduced with AAV2-shCtrl or AAV2-shTppp3 vectors. Quantification of mean neurite length per cell after transduction showed that Tppp3 knockdown decreases neurite outgrowth by ~ 20%. Scale bar = 100 µm. Each data point reflects an independent cell culture. Statistical significance was determined using one sample t-test (****p < 0.0001, ***p < 0.001). Mean ± SEM is shown

Fig. 3

Tppp3 promotes axon regeneration and improves RGC survival. A Scheme of experimental setup for ONC and sample collection. AAV2 vectors were intravitreally injected into eyes two weeks before ONC. Two days before sample collection, CTB-555, an anterograde tracer, was intravitreally injected into eyes to label regenerating axons. Optic nerves and retinas were collected two weeks after ONC. B Representative images of optic nerve sections transduced with AAV2 vectors and labeled with CTB-555. Tppp3 overexpression significantly increases CTB-555+ axons two weeks after ONC. The crush site is marked with an asterisk. The optic nerve's proximal end (towards the eye globe) is located on the left, and the distal end (towards the brain) is on the right. Scale bar = 100 μm. C Quantification of the number of CTB-555+ axons at varying distances from the crush site after transduction with AAV2-control (n = 6 optic nerves) or AAV2-Tppp3-OE (n = 8 optic nerves). Tppp3 overexpression improves RGC regeneration at short distances from the crush site. Statistical significance was determined using an unpaired student’s t-test for each distance (**p < 0.01). Mean ± SEM is shown. D Representative images of RBPMS+ cells in flatmount retinas of (D i) negative controls without AAV transduction and ONC, or transduced with (D ii) AAV-CMV or (D iii) AAV-Tppp3-OE. Scale bar = 50 μm. E Tppp3 overexpression improves RGC survival following ONC. Quantification of the mean number of RBPMS+ RGCs in flatmount retinas after transduction with control AAV2-CMV or AAV2-Tppp3-OE, compared to the negative control without AAV2 transduction and ONC (n = 6 retinas). Statistical significance was determined using an unpaired student’s t-test (****p < 0.0001, **p < 0.01). Mean ± SEM is shown. F TPPP3 protein expression is significantly decreased in the optic nerve after ONC compared to sham control

Fig. 4

Tppp3 overexpression increases Bmp4 and inflammation-related genes expression. A, C Several genes related to axon regeneration and survival were upregulated after transduction of AAV2-Tppp3-OE in the whole retina two days after ONC. B Real-time qPCR data comparing changes in mRNA gene expression (relative to Gapdh housekeeping gene) showed significantly increased Bmp4 expression two days after ONC (n = 3 retinas). Statistical significance was determined using an unpaired student’s t-test (***p < 0.001). Mean ± SEM is shown. D Identified GO terms that highlight terms related to inflammation and BMP signaling increase after Tppp3 overexpression

Fig. 5

Model of Tppp3’s role in CNS axonal regeneration. Graphic created with BioRender.com