Long-Term Effects of Neural Precursor Cell Transplantation on Secondary Injury Processes and Functional Recovery after Severe Cervical Contusion-Compression Spinal Cord Injury

Cervical spinal cord injury (SCI) remains a devastating event without adequate treatment options despite decades of research. In this context, the usefulness of common preclinical SCI models has been criticized. We, therefore, aimed to use a clinically relevant animal model of severe cervical SCI to assess the long-term effects of neural precursor cell (NPC) transplantation on secondary injury processes and functional recovery. To this end, we performed a clip contusion-compression injury at the C6 level in 40 female Wistar rats and a sham surgery in 10 female Wistar rats. NPCs, isolated from the subventricular zone of green fluorescent protein (GFP) expressing transgenic rat embryos, were transplanted ten days after the injury. Functional recovery was assessed weekly, and FluoroGold (FG) retrograde fiber-labeling, as well as manganese-enhanced magnetic resonance imaging (MEMRI), were performed prior to the sacrifice of the animals eight weeks after SCI. After cryosectioning of the spinal cords, immunofluorescence staining was conducted. Results were compared between the treatment groups (NPC, Vehicle, Sham) and statistically analyzed (p < 0.05 was considered significant). Despite the severity of the injury, leading to substantial morbidity and mortality during the experiment, long-term survival of the engrafted NPCs with a predominant differentiation into oligodendrocytes could be observed after eight weeks. While myelination of the injured spinal cord was not significantly improved, NPC treated animals showed a significant increase of intact perilesional motor neurons and preserved spinal tracts compared to untreated Vehicle animals. These findings were associated with enhanced preservation of intact spinal cord tissue. However, reactive astrogliosis and inflammation where not significantly reduced by the NPC-treatment. While differences in the Basso–Beattie–Bresnahan (BBB) score and the Gridwalk test remained insignificant, animals in the NPC group performed significantly better in the more objective CatWalk XT gait analysis, suggesting some beneficial effects of the engrafted NPCs on the functional recovery after severe cervical SCI.

Insights Into the Biological Role of NEDD4L E3 Ubiquitin Ligase in Human Cancers

Neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) is an E3 ubiquitin ligase that has been reported to participate in multiple cellular procedures by regulating of substrate ubiquitination and subsequent protein degradation. A great amount of evidence has demonstrated that NEDD4L mainly functions as a tumor suppressor in most cancer types, while it also acts as an oncogene in a few cancers. In this review, we summarize the potential role of NEDD4L in carcinogenesis and the related underlying molecular mechanism to improve our understanding of its functions in the tumorigenesis of human malignancies. Developing clinical drugs targeting NEDD4L could be a potential therapeutic strategy for cancer therapy in the future.

The Role of Mcl-1 in Embryonic Neural Precursor Cell Apoptosis

Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, regulates neural precursor cell (NPC) survival in both the developing and adult mammalian nervous system. It is unclear when during the neurogenic period Mcl-1 becomes necessary for NPC survival and whether Bax is the sole pro-apoptotic target of Mcl-1. To address these questions, we used the nervous system-specific Nestin-Cre Mcl-1 conditional knockout mouse line (Mcl-1 CKO) to assess the anti-apoptotic role of Mcl-1 in developmental neurogenesis. Loss of Mcl-1 resulted in a wave of apoptosis beginning in the brainstem and cervical spinal cord at embryonic day 9.5 (E9.5) and in the forebrain at E10.5. Apoptosis was first observed ventrally in each region and spread dorsally over time. Within the spinal cord, apoptosis also spread in a rostral to caudal direction following the path of differentiation. Breeding the Mcl-1 CKO mouse with the Bax null mouse rescued the majority of NPC from apoptosis except in the dorsomedial brainstem and ventral thoracic spinal cord where only 50% were rescued. This demonstrates that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis. Interestingly, although co-deletion of Bax rescued the majority of NPC apoptosis, it resulted in embryonic lethality at E13, whereas conditional deletion of both Mcl-1 and Bax rescued embryonic lethality. In summary, this study demonstrates the widespread dependency on Mcl-1 during nervous system development.

Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

Targeting neddylation E2s: a novel therapeutic strategy in cancer

Ubiquitin-conjugating enzyme E2 M (UBE2M) and ubiquitin-conjugating enzyme E2 F (UBE2F) are the two NEDD8-conjugating enzymes of the neddylation pathway that take part in posttranslational modification and change the activity of target proteins. The activity of E2 enzymes requires both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of neural precursor cell-expressed developmentally downregulated 8 (NEDD8). By recruiting E3 ligases and targeting cullin and non-cullin substrates, UBE2M and UBE2F play diverse biological roles. Currently, there are several inhibitors that target the UBE2M-defective in cullin neddylation protein 1 (DCN1) interaction to treat cancer. As described above, this review provides insights into the mechanism of UBE2M and UBE2F and emphasizes these two E2 enzymes as appealing therapeutic targets for the treatment of cancers.

Inhibiting Neddylation with MLN4924 Suppresses Growth and Delays Multicellular Development in Dictyostelium discoideum

Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.