Copper homeostasis and the ubiquitin proteasome system.

Copper is involved in many physiological pathways and important biological processes as a cofactor of several copper-dependent enzymes. Given the requirement for copper and its potential toxicity, intracellular copper levels are tightly controlled. Disturbances of human copper homeostasis are characterized by disorders of copper overload (Wilson's disease) or copper deficiency (Menkes disease). The maintenance of cellular copper levels involves numerous copper transporters and copper chaperones. Recently, accumulating evidence has revealed that components of the ubiquitin proteasome system (UPS) participate in the posttranslational regulation of these proteins, suggesting that they might play a role in maintaining copper homeostasis. Cellular copper levels could also affect the activity of the UPS, indicating that copper homeostasis and the UPS are interdependent. Copper homeostasis and the UPS are essential to the integrity of normal brain function and while separate links between neurodegenerative diseases and UPS inhibition/copper dyshomeostasis have been extensively reported, there is growing evidence that these two networks might contribute synergistically to the occurrence of neurodegenerative diseases. Here, we review the role of copper and the UPS in the development of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, and discuss the genetic interactions between copper transporters/chaperones and components of the UPS.

Human Menstrual Blood-Derived Endometrial Stem Cells Promote Functional Recovery by Improving the Inflammatory Microenvironment in a Mouse Spinal Cord Injury Model.

Spinal cord injury (SCI) is a traumatic injury of the central nervous system. Because neurons are damaged and difficult to regenerate after SCI, its repair remains challenging. However, recent research on stem cell therapy have favored its use after SCI. In this study, based on the establishment of a mouse SCI model, human menstrual blood-derived endometrial stem cells (MenSCs) were intrathecally injected to explore the role and molecular mechanism of MenSCs in SCI. MenSCs were transplanted following SCI in the animal model, and behavioral evaluations showed that MenSC transplantation improved functional recovery. Therefore, samples were collected after 7 days, and transcriptome sequencing was performed. Gene Ontology (GO) enrichment analysis revealed that SCI is closely related to immune system processes. After transplantation of MenSCs, the immune response was significantly activated. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, MenSC transplantation was found to be closely related to Th1, Th2, and Th17 cell differentiation pathways. Neuronal damage and glial cell proliferation and activation in the different groups were detected by fluorescence immunohistochemistry and Western blotting 7 days after SCI. Simultaneously, the activation of different types of microglia was detected and the expression of pro-inflammatory and anti-inflammatory factors was quantitatively analyzed. The results showed that MenSC transplantation and sonic hedgehog (Shh)-induced MenSCs accelerated neuronal recovery at the injured site, inhibited the formation of glial cells and microglial activation at the injured site, inhibited the expression of inflammatory factors, and improved the inflammatory microenvironment to achieve functional recovery of SCI. This study provides an experimental basis for the study of the role and molecular mechanism of MenSCs in SCI repair, and a reference for the role of Shh-induced MenSCs in SCI repair.

The Vhl E3 ubiquitin ligase complex regulates melanisation via sima, cnc and the copper import protein Ctr1A.

VHL encodes a tumour suppressor, which possesses E3 ubiquitin ligase activity in complex with EloC and Cul2. In tumour cells or in response to hypoxia, VHL activity is lost, causing accumulation of the transcription factor HIF-1alpha. In this study, we demonstrated that in Drosophila, Rpn9, a regulatory component of the 26 s proteasome, participates in the Vhl-induced proteasomal degradation of sima, the Drosophila orthologue of HIF-1alpha. Knockdown of Vhl induces increased melanisation in the adult fly thorax and concurrent decrease in pigmentation in the abdomen. Both these defects are rescued by knockdown of sima and partially by knockdown of cnc, which encodes the fly orthologue of the transcription factor Nrf2, the master regulator of oxidative stress response. We further show that sima overexpression and Rpn9 knockdown both result in post-translational down-regulation of the copper uptake transporter Ctr1A in the fly eye and that Ctr1A expression exacerbates Vhl knockdown defects in the thorax and rescues these defects in the abdomen. We conclude that Vhl negatively regulates both sima and cnc and that in the absence of Vhl, these transcription factors interact to regulate Ctr1A, copper uptake and consequently melanin formation. We propose a model whereby the co-regulatory relationship between sima and cnc flips between thorax and abdomen: in the thorax, sima is favoured leading to upregulation of Ctr1A; in the abdomen, cnc dominates, resulting in the post-translational downregulation of Ctr1A.

The E3 ubiquitin ligase Slimb/ß-TrCP is required for normal copper homeostasis in Drosophila.

The Drosophila Slimb (Slmb) gene encodes a Skp1-Cul1-F-box (SCP) E3 ubiquitin ligase orthologous to the human ß-TrCP/BTRC protein. Slmb and/or BTRC play regulatory roles in numerous biological processes by ubiquitinating several substrate proteins which are then targeted for proteasomal degradation. Here, we demonstrate an additional role for Slmb in maintaining cellular copper homeostasis. In the thorax, midgut and eye, Slmb knockdown causes copper deficiency phenotypes which can be rescued by increasing cellular copper levels via decreased efflux or increased uptake. Furthermore, Slmb knockdown results in decreased levels of the copper transporters Ctr1A and ATP7, indicating Slmb is required to regulate copper homeostasis. We also present evidence that the transcription factor Cap-n-Collar (Nrf2 in mammals), a known substrate of Slmb/BTRC, mediates Slmb's regulatory effect on Ctr1A in a post-transcriptional manner.

The Mechanism of Rap1 Regulates N-cadherin to Control Neuronal Migration.

Rap1 and N-cadherin regulate glia-independent translocation of cortical neurons. It remains unclear how Rap1 regulates N-cadherin-mediated neuronal migration. Here, we overexpressed Rap1gap in mouse brains (embryonic day 16) to inactivate Rap1, and observed that neurons did not migrate to the outer layer. We confirmed that Rap1 was involved in the regulation of late neurons in vivo. Rap1gap overexpression and Rap1 suppression in CHO cells decreased the expression of cytoskeletal proteins such as tubulin. Changes in the expression of cell morphology regulators, such as N-cadherin and ß-catenin, were also observed. Inhibition of N-cadherin in mouse brains prevented neuronal migration to the outer layer. The morphology of CHO cells was changed after overexpression of Rap1gap. We propose that Rap1 regulates the expression of N-cadherin during embryonic development, which affects ß-catenin expression. Beta-catenin in turn regulates cytoskeletal protein expression, ultimately affecting neuronal morphology and migration.

Wnt3a Ectopic Expression Interferes Axonal Projection and Motor Neuron Positioning During the Chicken Spinal Cord Development.

The formation of dorsal-ventral axis of the spinal cord is controlled largely by dorsal signals such as Wnts (which are members of the wingless + MMTV integrants, Int family), besides ventral signals such as sonic hedgehog (Shh). Wnt3a, one of the Wnt family members, is involved in multiple cellular functions, including self-renewal, proliferation, differentiation, and motility. Here, we aim to study the mechanism of the regulation of chicken spinal cord patterning by Wnt3a. In this study, Wnt3a was ectopically expressed in the spinal cord of developing chicken embryos by in ovo electroporation. The results of immunofluorescent staining revealed that Wnt3a ectopic expression caused the abnormality of commissural axonal projection and the formation of nerve fibers was interrupted. It is worth noting that neurons in the ventricular zone, especially motor neurons, could not migrate laterally after the Wnt3a overexpression, which led to the malformation of motor column. In addition, we found that neurons could not protrude axons outwardly after overexpression of Wnt3a in the spinal cord. It was also found that Wnt3a overexpression inhibited the outgrowth of processes in culturing SH-SY5Y cells. In conclusion, we proposed that Wnt3a regulates neuronal morphology, which subsequently disrupts axonal projection and motor neuron positioning during spinal cord development.

Combined use of in ovo electroporation and cultured neurons for gene function analysis of embryogenesis in the chicken optic tectum.

Chicken embryos are used widely in the fields of developmental biology and neurobiology. The chicken embryo also serves as a model to analyze gene expression and function using in ovo electroporation. Plasmids may be injected into the spinal cord or tectum of the chicken central nervous system by microinjection for electroporation. Here, we developed a novel method that combines in ovo electroporation and neuronal culturing to study gene function in the chicken tectum during embryo development. Our method can be used to study in-vivo and in-vitro exogenous genes' function. In addition, live cell imaging microscopy, immunostaining, and transfection can be used with our method to study neuronal growth, development, neurite growth and retraction, and axonal pathfinding. Our result showed that axons were present in isolated neurons after culturing for 24 h, and cell debris was low after replacing the media at 48 h. Many GFP-expressing neurons were observed in the cultured cells after 48 h. We successfully cultured the neurons for 3 weeks. Together, this method combines in ovo electroporation and neuronal culturing advantages and is more convenient for the gene function analysis.

Telocytes: a potential defender in the spleen of Npc1 mutant mice.

Niemann-Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1-/- ), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1-/- mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c-Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1-/- mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1-/- spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.