MiR-155-5p Aggravated Astrocyte Activation and Glial Scarring in a Spinal Cord Injury Model by Inhibiting Ndfip1 Expression and PTEN Nuclear Translocation.

Central nervous injury and regeneration repair have always been a hot and difficult scientific questions in neuroscience, such as spinal cord injury (SCI) caused by a traffic accident, fall injury, and war. After SCI, astrocytes further migrate to the injured area and form dense glial scar through proliferation, which not only limits the infiltration of inflammatory cells but also affects axon regeneration. We aim to explore the effect and underlying mechanism of miR-155-5p overexpression promoted astrocyte activation and glial scarring in an SCI model. MiR-155-5p mimic (50 or 100 nm) was used to transfect CTX-TNA2 rat brain primary astrocyte cell line. MiR-155-5p antagonist and miR-155-5p agomir were performed to treat SCI rats. MiR-155-5p mimic dose-dependently promoted astrocyte proliferation, and inhibited cell apoptosis. MiR-155-5p overexpression inhibited nuclear PTEN expression by targeting Nedd4 family interacting protein 1 (Ndfip1). Ndfip1 overexpression reversed astrocyte activation which was induced by miR-155-5p mimic. Meanwhile, Ndfip1 overexpression abolished the inhibition effect of miR-155-5p mimic on PTEN nuclear translocation. In vivo, miR-155-5p silencing improved SCI rat locomotor function and promoted astrocyte activation and glial scar formation. And miR-155-5p overexpression showed the opposite results. MiR-155-5p aggravated astrocyte activation and glial scarring in a SCI model by targeting Ndfip1 expression and inhibiting PTEN nuclear translocation. These findings have ramifications for the development of miRNAs as SCI therapeutics.

Research progress on the role of Ndfip1 (Nedd4 family interacting protein 1) in immune cells.

Nedd4 family interacting protein 1 (Ndfip1) was first mentioned in an article in 2000. Since its discovery, related studies have shown that this protein is associated with apoptosis, neuroprotection, substance transport, ubiquitination, and immune regulation. It is noteworthy that the lack of Ndfip1 can lead to death in fetal mice. Researchers generally believe that the function of Ndfip1 is closely related to individual immune capacity and have published a large number of articles. However, a comprehensive classification of the immune regulatory function of Ndfip1 is still lacking. In this review, we will overview and discuss this new perspective, focusing on the role of Ndfip1 in the proliferation, differentiation, and cell activity of CD4+ T cells, CD8+ T cells, mast cells, and eosinophils. This review provides an updated summary of Ndfip1, which will unveil novel therapeutic targets. Finally, the conclusion is that Ndfip1 mainly plays a negative regulatory role in immune cells by maintaining the stability of the immune response and limiting its overexpression.

Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases.

NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1-related ubiquitin ligases.

Nicotine promotes tumor progression and epithelial-mesenchymal transition by regulating the miR-155-5p/NDFIP1 axis in pancreatic ductal adenocarcinoma.

BACKGROUND: Nicotine, the major component of cigarette smoke, has been reported to promote pancreatic ductal adenocarcinoma (PDAC) growth and invasion. Deregulation of microRNA (miRNA) expression is found in many cancers, including PDAC. The effects of nicotine on miRNAs change in PDAC progression remain unknown. METHODS: The effects of cigarette smoking/nicotine exposure on PDAC cell lines and tissues were evaluated. Quantitative real-time PCR and in situ hybridization assays were used to determine miR-155-5p expression in human PDAC tissue and cell lines upon cigarette smoking/nicotine exposure. Bioinformatics, loss-of-function experiments, luciferase reporter assay were performed to validate Nedd4 family interacting protein 1 (NDFIP1) as a direct target of miR-155-5p. The potentials of systemic miR-155-5p inhibitor-based therapy in overcoming nicotine exposure were evaluated in tumor xenograft model. RESULTS: Nicotine promoted PDAC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in a dose-response manner. MiR-155-5p was found to be highly expressed in PDAC cell lines and tissues upon cigarette smoking/nicotine exposure. Functional studies showed that miR-155-5p knockdown could override the enhancement of oncogenic activity due to nicotine exposure in vitro and in vivo by directly interacting with the 3' untranslated regions (UTRs) of NDFIP1. CONCLUSIONS: These data demonstrate that nicotine-regulated miR-155-5p/NDFIP1 promotes tumor progression and EMT of PDAC.

Allosteric auto-inhibition and activation of the Nedd4 family E3 ligase Itch.

The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.

PTEN secretion in exosomes.

PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis.

Nedd4 family interacting protein 1 (Ndfip1) promotes death of pancreatic beta cells.

High-throughput siRNA screening was employed to identify novel genes that regulate cytokine-induced death of pancreatic ß-cells. One of the 'hits' was Nedd4 family interacting protein 1 (Ndfip1), an adaptor and activator of Nedd4-family ubiquitin ligases. Silencing of Ndfip1 inhibited cytokine-induced apoptosis of mouse and human pancreatic islets and promoted glucose-stimulated insulin secretion. These effects were associated with an increase in the cellular content of JunB, a potent inhibitor of ER stress and apoptosis. Silencing of Ndfip1 also increased the expression of ATF4, IRE-1α, and the spliced form of XBP that govern the unfolded protein response (UPR) and relieve cytokine-induced ER stress, while overexpression of Ndfip1 exerted opposite effects. These findings implicate Ndfip1 in the degradation of JunB; inhibition of the UPR and insulin secretion; and promotion of cytokine-induced death of pancreatic ß-cells.

NDFIP1 limits cellular TAZ accumulation via exosomal sorting to inhibit NSCLC proliferation.

NDFIP1 has been previously reported as a tumor suppressor in multiple solid tumors, but the function of NDFIP1 in NSCLC and the underlying mechanism are still unknown. Besides, the WW domain containing proteins can be recognized by NDFIP1, resulted in the loading of the target proteins into exosomes. However, whether WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) can be packaged into exosomes by NDFIP1 and if so, whether the release of this oncogenic protein via exosomes has an effect on tumor development has not been investigated to any extent. Here, we first found that NDFIP1 was low expressed in NSCLC samples and cell lines, which is associated with shorter OS. Then, we confirmed the interaction between TAZ and NDFIP1, and the existence of TAZ in exosomes, which requires NDFIP1. Critically, knockout of NDFIP1 led to TAZ accumulation with no change in its mRNA level and degradation rate. And the cellular TAZ level could be altered by exosome secretion. Furthermore, NDFIP1 inhibited proliferation in vitro and in vivo, and silencing TAZ eliminated the increase of proliferation caused by NDFIP1 knockout. Moreover, TAZ was negatively correlated with NDFIP1 in subcutaneous xenograft model and clinical samples, and the serum exosomal TAZ level was lower in NSCLC patients. In summary, our data uncover a new tumor suppressor, NDFIP1 in NSCLC, and a new exosome-related regulatory mechanism of TAZ.

Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch.

JAK1 depletion or downregulation was previously reported to account for coronavirus inhibition. Here, we found that AG1024, an IR (insulin receptor) and IGF-1R (insulin-like growth factor 1 receptor) inhibitor, diminishes JAK1 protein levels and exerts anti-coronaviral activities with EC50 values of 5.2 ± 0.3 µM against transmissible gastroenteritis coronavirus (TGEV) and 4.3 ± 0.3 µM against human flu coronavirus OC43. However, although the IR and IGF-1R signaling pathways are activated by insulin or IGF-1 in swine testis cells, they are not triggered upon TGEV infection. AG1024, therefore, inhibits coronaviral replication and downregulates JAK1 protein levels independently of IR and IGF-1R. Moreover, JAK1 proteolysis caused by AG1024 was found through activation of upstream Ndfip1/2 and its effector NEDD4-like E3 ligase Itch. In addition, ouabain, which was reported to mediate JAK1 proteolysis causing anti-coronaviral activity by activation of Ndfip1/2 and NEDD4 E3 ligase, additively inhibited anti-coronaviral activity and JAK1 diminishment in combination with AG1024. This study provides novel insights into the pharmacological effects of AG1024 and Itch E3 ligase mediated JAK1 proteolysis and identified Ndfip1/2 as a cognate effector for JAK1 proteolysis via the diversified E3 ligases NEDD4 and NEDD4-like Itch. These findings are expected to provide valued information for the future development of anti-viral agents.

Ndfip1 Prevents Rotenone-Induced Neurotoxicity and Upregulation of α-Synuclein in SH-SY5Y Cells.

Nedd4 family interacting protein 1 (Ndfip1) is an adaptor of Nedd4-family ubiquitin ligases. Experimental results showed that Ndfip1 had a potential neuroprotective effect in neurology diseases. However, the neuroprotective effect and the underlying mechanisms of Ndfip1 in Parkinson's disease (PD) have not yet been fully elucidated. Therefore, in this study, we explored the neuroprotective effect of Ndfip1 against mitochondrial complex I inhibitor rotenone in a human dopaminergic neuroblastoma SH-SY5Y cell line and further elucidated its possible underlying mechanisms. Our results showed that rotenone could induce the up-regulation of α-synuclein (α-syn) in both mRNA and protein levels. The expression of Ndfip1 decreased at 24 h after rotenone treatment. Further study showed that high expression of Ndfip1 could protect SH-SY5Y cells against rotenone-induced neurotoxicity and antagonize the rotenone-induced increase in α-syn protein levels. In addition, high expression of Ndfip1 inhibited rotenone-induced increase in the protein levels of caspase-3 and decrease in tyrosine hydroxylase (TH). Further study showed that Ndfip1 did not affect the protein expression of iron regulatory protein 1 (IRP1), transferrin receptor 1 (TfR1), while antagonized the increase in protein levels of P62 and ferritin L caused by rotenone. Our findings provide specific identification of Ndfip1 proteins to inhibit the increase of α-syn in rotenone-induced SH-SY5Y cells. Ndfip1 might be a new theoretical drug target for the prevention and treatment of PD.

Oxysophocarpine protects airway epithelial cells against inflammation and apoptosis by inhibiting miR-155 expression.

Oxysophocarpine (OSC) has been documented for anti-inflammatory activity. However, the mechanisms of OSC in anti-inflammation are unclear. Aim: To investigate the protective effects of OSC on inflammation and apoptosis induced by lipopolysaccharide in NCI-H292 and human primary airway epithelial cells. Materials & methods: MTT and Annexin V-FITC/PI staining were used to detect cells viability. Inflammatory responses were determined by ELISA. The quantitative real-time PCR (qRT-PCR) and western blot were used to detect mRNA/miRNA and protein expressions respectively. Co-immunoprecipitation was investigated for protein interactions. Results & conclusion: miR-155 mimics significantly induced cell apoptosis, inflammatory responses and MAPK and NF-κB pathways. NDFIP1 was identified as the target of miR-155. OSC protected cells against apoptosis and inflammatory responses and compromised miR-155 activity by attenuating MAPK and NF-κB pathways.

The miR-873/NDFIP1 axis promotes hepatocellular carcinoma growth and metastasis through the AKT/mTOR-mediated Warburg effect.

Hepatocellular carcinoma (HCC) progression depends on cellular metabolic reprogramming as both direct and indirect consequence of oncogenic lesions. However, the underlying mechanisms are still understood poorly. Here, we report that miR-873 promotes Warburg effect in HCC cells by increasing glucose uptake, extracellular acidification rate (ECAR), lactate production, and ATP generation, and decreasing oxygen consumption rate (OCR) in HCC cells. Mechanistically, we show that miR-873 activates the key glycolytic proteins AKT/mTOR via targeting NDFIP1 which triggers metabolic shift. We further demonstrate that enhanced glycolysis is essential for the role of miR-873 to drive HCC progression. By using immunohistochemistry analysis, we show a link between the aberrant expression of miR-873, NDFIP1, and phospho-AKT in clinical HCC samples. We also found that miR-873 was up-regulated by HIF1α, a critical glycolysis-related transcription factor. However, BAY 87-2243, a HIF1α specific inhibitor, blocks miR-873 mediated tumor growth and metastasis in nude mice. Collectively, our data uncover a previously unappreciated function of miR-873 in HCC cell metabolism and tumorigenesis, suggesting that targeting miR-873/NDFIP1 axis could be a potential therapeutic strategy for the treatment of HCC patients.

chi-miR-143-3p Promotes Apoptosis of Mammary Gland Epithelial Cells from Dairy Goats by Targeting Ndfip1.

The mammary gland is an important organ for lactation in dairy goats. Mammary gland development and lactation functions are primarily regulated by natural hormones and certain crucial regulatory factors. Nedd4 family-interacting protein 1 (Ndfip1) can specifically bind to neural precursor cell-expressed, developmentally downregulated protein 4 (Nedd4) family members to participate in ubiquitination, which in turn regulates a range of biological processes in the body. However, the effects of Ndfip1 expression regulation at the post-transcriptional level on the development of mammary gland cells have not been previously reported. To study the regulation of Ndfip1 at post-transcriptional level, the overexpression and interference vectors of Ndfip1 were constructed, and co-transfected into the primary mammary gland epithelial cells cultured in vitro with miR-143 mimics and inhibitor. Dual luciferase reporter gene system, real-time quantitative polymerase chain reaction, western blotting, cholecystokinin octapeptide assays, and flow cytometry were used to identify their regulation and function. As a result, Ndfip1 was targeted and regulated by miR-143, which influences the development of mammary gland epithelial cells in dairy goats cultured in vitro. This study will lay an experimental foundation for further understanding the functions of Ndfip1 and miR-143.

Ubiquitin Regulation of Trk Receptor Trafficking and Degradation.

The regulation of Trk receptors is critical for orchestrating multiple signalling pathways required for developing and maintaining neuronal networks. Activation of Trk receptors results in signalling, internalisation and subsequent degradation of the protein. Although ubiquitination of TrkA by Nedd4-2 has been identified as an important degradation pathway, much less is known about the pathways regulating the degradation of TrkB and TrkC. Critical to the interaction between TrkA and Nedd4-2 is a PPxY motif present within TrkA but absent in TrkB and TrkC. Given the absence of this interaction motif, it remains to be determined how TrkB and TrkC are ubiquitinated. Here we report that the adaptor protein Ndfip1 can interact with all three Trk receptors and show for TrkB the recruitment of Nedd4-2 through PPxY motifs present in Ndfip1. Ndfip1 mediates the ubiquitination of TrkB, resulting in receptor trafficking predominantly on Rab7 containing late endosomes, highlighting a pathway for TrkB degradation at the lysosome. In vitro, overexpression of Ndfip1 increased TrkB ubiquitination and decreased viability of BDNF-dependent primary neurons. In vivo, conditional genetic deletion of Ndfip1 increased TrkB in the brain and resulted in enlargement of the granular cell layer of the dentate gyrus.

The Ubiquitin Ligase Adaptor NDFIP1 Selectively Enforces a CD8+ T Cell Tolerance Checkpoint to High-Dose Antigen.

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.

Research progress on the role of Ndfip1 (Nedd4 family interacting protein 1) in immune cells

Nedd4 family interacting protein 1 (Ndfip1) was first mentioned in an article in 2000. Since its discovery, related studies have shown that this protein is associated with apoptosis, neuroprotection, substance transport, ubiquitination, and immune regulation. It is noteworthy that the lack of Ndfip1 can lead to death in fetal mice. Researchers generally believe that the function of Ndfip1 is closely related to individual immune capacity and have published a large number of articles. However, a comprehensive classification of the immune regulatory function of Ndfip1 is still lacking. In this review, we will overview and discuss this new perspective, focusing on the role of Ndfip1 in the proliferation, differentiation, and cell activity of CD4+ T cells, CD8+ T cells, mast cells, and eosinophils. This review provides an updated summary of Ndfip1, which will unveil novel therapeutic targets. Finally, the conclusion is that Ndfip1 mainly plays a negative regulatory role in immune cells by maintaining the stability of the immune response and limiting its overexpression (AU)

Lower Expression of Ndfip1 Is Associated With Alzheimer Disease Pathogenesis Through Decreasing DMT1 Degradation and Increasing Iron Influx.

We have previously reported that high expression of divalent metal transporter 1 (DMT1) plays a crucial role in iron dyshomeostasis and ß-amyloid (Aß) peptide generation in the brain of Alzheimer's disease (AD). Recent studies have shown that Nedd4 family interacting protein 1 (Ndfip1) can degrade DMT1 through ubiquitination pathway and reduce the accumulation of intracellular iron. The present study aims to evaluate whether Ndfip1 is involved in AD pathogenesis through mediating DMT1 degradation and iron metabolism. ß-amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mouse and Ndfip1 transfected SH-SY5Y cells were used in this study. Immunohistochemistry and Western blot were performed to examine the distribution and expression levels of Ndfip1 and DMT1. In addition, ELISA and calcein fluorescence were carried out for analyzing the levels of Aß peptide and iron influx, respectively. The results showed that Ndfip1 immunoreactivity was decreased in the cortex and hippocampus of APP/PS1 mice, compared with wild type (WT) controls. Colocalization of Ndfip1 and Aß within senile plaques could be observed. Immunoblot analyses showed that low expression of Ndfip1 and high expression of DMT1 proteins were detected in APP/PS1 mouse brain, compared with age-matched WT animals. Overexpression of Ndfip1 down-regulated DMT1 expression, and reduced iron influx and Aß secretion in SH-SY5Y cells. Further, overexpressed Ndfip1 significantly attenuated iron-induced cell damage in Ndfip1 transfected cells. The present study suggests that lower expression of Ndfip1 might be associated with the pathogenesis of AD, through decreasing DMT1 degradation and increasing iron accumulation in the brain.

MiR-155 Promotes Uveal Melanoma Cell Proliferation and Invasion by Regulating NDFIP1 Expression.

MicroRNAs refer to small RNA molecules that destroy the messenger RNA by binding on them inhibiting the production of protein. However, the role of miR-155 in uveal melanoma metastasis remains largely unknown. In this study, we found that miR-155 was upregulated in both uveal melanoma cells and tissues. Transfection of miR-155 mimic into uveal melanoma cells led to an increase in cell growth and invasion; in contrast, inhibition of miR-155 resulted in opposite effects. Also, we identified Nedd4-family interacting protein 1 as a direct target of miR-155, and the expression of Nedd4-family interacting protein 1 was inhibited by miR-155. Furthermore, ectopic expression of Nedd4-family interacting protein 1 restored the effects of miR-155 on cell proliferation and invasion of uveal melanoma cells. In conclusion, miR-155 acts as a tumor promotor in uveal melanoma through increasing cell proliferation and invasion. Thus, miR-155 might serve as a potential therapeutic target in patients with uveal melanoma.

Protective effects of Ndfip1 on MPP(+)-induced apoptosis in MES23.5 cells and its underlying mechanisms.

Apoptosis has been implicated as one of the important mechanisms involved in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Increasing evidence suggests that Ndfip1 is a neuroprotective protein, and Ndfip1-mediated protein ubiquitination might be a possible survival strategy in neuronal injury. The aim of the present study is to investigate the neuroprotective effect of Ndfip1 on 1-methyl-4-phenylpyridinium (MPP(+))-treated MES23.5 cells and the underlying mechanisms. Results showed that overexpression of Ndfip1 could significantly attenuate MPP(+)-induced cell loss and nuclear condensation. Further experiments demonstrated that Ndfip1 could increase Bcl-2/Bax ratio, suppress cytochrome c release from the mitochondria to cytoplasm and decrease caspase-3 activation induced by MPP(+). These results suggested that Ndfip1 protected MES23.5 cells against MPP(+) by its anti-apoptotic effect. In addition, we found that Ndfip1 overexpression could decrease the protein level of dopamine transporter (DAT). In parallel, proteasome inhibitor MG132 could markedly reverse Ndfip1-induced degradation of DAT. These data suggest that Ndfip1 exerts its inhibitory effect on DAT by modulating DAT degradation, in which ubiquitin-proteasome system activation might be involved. Collectively, our study indicated that the ability to decrease the DAT of Ndfip1 might be one of the mechanisms underlying its protective effect on MPP(+)-induced cell damage in MES23.5 cells.

Ndfip1 attenuated 6-OHDA-induced iron accumulation via regulating the degradation of DMT1.

Elevated iron levels and increased expression of divalent metal transporter 1 (DMT1) in the substantia nigra of Parkinson's disease (PD) have been reported. Nedd4 family-interacting protein 1 (Ndfip1), an adaptor protein for the Nedd4 family of ubiquitin ligases, played an essential role in regulating DMT1 and iron homeostasis in human cortical neurons. In this study, we demonstrated that the expression of Ndfip1 decreased in 6-hydroxydopamine (6-OHDA)-induced PD rats and 6-OHDA-treated MES23.5 dopaminergic cells. Further study showed that the decrease of Ndfip1 occurred earlier than the increase of DMT1 with iron-responsive element (DMT1 + IRE) in 6-OHDA-treated MES23.5 cells, indicating that the decrease of Ndfip1 might be involved in the increase of DMT1 + IRE. In addition, we demonstrated that overexpression of Ndfip1 caused DMT1 + IRE downregulation, resulting in the decreased iron influx and iron-induced neurotoxicity. Although Ndfip1 knockdown led to decreased protein levels of DMT1 + IRE, partially aggravated iron-induced neurotoxicity. Further experiments showed that 6-OHDA-induced decrease in Ndfip1 levels might be related to proteasomal and lysosomal activations and oxidative stress caused by 6-OHDA. These data suggest that decreased Ndfip1 expression might contribute to the pathogenesis of 6-OHDA-induced iron accumulation and Ndfip1 could attenuate 6-OHDA-induced iron accumulation via regulating the degradation of DMT1.