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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.

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.

During development, protein substrates need to be removed and degraded when they are no longer required. The E3 ubiquitin ligases, including Nedd4 family proteins, are a major group of enzymes responsible for adding ubiquitin chains to protein substrates prior to their degradation. Ndfip1 (Nedd4 family-interacting protein 1) is an adaptor and activator for Nedd4-family ubiquitin ligases for increasing substrate specificity. To study Nedd4-mediated ubiquitination during cortical development, we have mapped the spatio-temporal dynamics of Ndfip1 protein expression by immunocytochemistry. Ndfip1 expression was observed from embryonic day 11 (E11.5) until adult stages. Its presence increased during the postnatal stages and peaked at postnatal day 7 (P7). Spatially, Ndfip1 was found in the ventricular and marginal zones during corticogenesis but also in the cortical plate and subplate during midstage cortical development (E15.5). Postnatally, Ndfip1 was expressed in all cortical neurons (but not in glial cells) and this expression was both ubiquitous and uniform across cortical layers involving both pyramidal and non-pyramidal neurons. This consistent but dynamic pattern of Ndfip1 expression in temporal and spatial domains of the cortical landscape is indicative of complex programs of protein ubiquitination during corticogenesis.

Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.

The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [Co(III)(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl(2) that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic Co(III) complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of Co(III) to Co(II) leading to release of Co(II) ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by Co(III) complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of Co(III) complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit.

Although brain-derived neurotrophic factor (BDNF) has been shown to promote peripheral myelination during development and remyelination after injury, the precise mechanisms mediating this effect remain unknown. Here, we determine that BDNF promotes myelination of nerve growth factor-dependent neurons, an effect dependent on neuronal expression of the p75 neurotrophin receptor, whereas BDNF inhibits myelination of BDNF-dependent neurons via the full-length TrkB receptor. Thus, BDNF exerts contrasting effects on Schwann cell myelination, depending on the complement of BDNF receptors that are expressed by different subpopulations of dorsal root ganglion neurons. These results demonstrate that BDNF exerts contrasting modulatory roles in peripheral nervous system myelination, and that its mechanism of action is acutely regulated and specifically targeted to neurons.

The spatial regulation of Pten is critical for its role as a tumour suppressor with both nuclear and cytoplasmic locations being implicated with distinct functions. In the cytoplasm, Pten plays a central role in opposing PI3K/Akt cell signalling, whereas in the nucleus, Pten is important for maintaining genome stability and enhancing the tumour suppressor activity of APC-CDH1. Despite this diversity in protein function at different subcellular locations, there is limited knowledge on how Pten is able to find different cellular niches. Here, we report that Rab5 GTPase is required for efficient trafficking and ubiquitination of Pten on endosomes inside the cytosol. Using bimolecular fluorescence complementation (BiFC) for imaging protein interactions, we observed that ubiquitinated Pten is localized to peri-nuclear and nuclear regions of the cell. Nuclear trafficking of Pten required both Rab5 as well as the E3 ligase adaptor protein Ndfip1. Rab5 colocalization with Pten was observed on endosomes and expression of a dominant negative form of Rab5 significantly reduced Pten ubiquitination and nuclear trafficking. Genomic deletion of Ndfip1 abrogated nuclear trafficking of ubiquitinated Pten, even in the presence of Rab5. Our findings show that endosomal trafficking and ubiquitination are important mechanisms for the subcellular distribution of Pten.

There is controversy whether accumulation of the tumor suppressor PTEN protein in the cell nucleus under stress conditions such as trauma and stroke causes cell death. A number of in vitro studies have reported enhanced apoptosis in neurons possessing nuclear PTEN, with the interpretation that its nuclear phosphatase activity leads to reduction of the survival protein phospho-Akt. However, there have been no in vivo studies to show that nuclear PTEN in neurons under stress is detrimental. Using a mouse model of injury, we demonstrate here that brain trauma altered the nucleo-cytoplasmic distribution of Pten, resulting in increased nuclear Pten but only in surviving neurons near the lesion. This event was driven by Ndfip1, an adaptor and activator of protein ubiquitination by Nedd4 E3 ligases. Neurons next to the lesion with nuclear PTEN were invariably negative for TUNEL, a marker for cell death. These neurons also showed increased Ndfip1 which we previously showed to be associated with neuron survival. Biochemical assays revealed that overall levels of Pten in the affected cortex were unchanged after trauma, suggesting that Pten abundance globally had not increased but rather Pten subcellular location in affected neurons had changed. Following experimental injury, the number of neurons with nuclear Pten was reduced in heterozygous mice (Ndfip1(+/-)) although lesion volumes were increased. We conclude that nuclear trafficking of Pten following injury leads to neuron survival not death.

Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease.

PTEN nuclear entry driven by ubiquitination is mediated by the ligase-interacting protein Ndfip1 and is essential for neuronal survival in mice after cerebral ischemia., PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family–interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1−/− fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.

The regulation of metal ion transport within neurons is critical for normal brain function. Of particular importance is the regulation of redox metals such as iron (Fe), where excess levels can contribute to oxidative stress and protein aggregation, leading to neuronal death. The divalent metal transporter 1 (DMT1) plays a central role in the regulation of Fe as well as other metals; hence, failure of DMT1 regulation is linked to human brain pathology. However, it remains unclear how DMT1 is regulated in the brain. Here, we show that DMT1 is regulated by Ndfip1 (Nedd4 family-interacting protein 1), an adaptor protein that recruits E3 ligases to ubiquitinate target proteins. Using human neurons we show the Ndfip1 is upregulated and binds to DMT1 in response to Fe and cobalt (Co) exposure. This interaction results in the ubiquitination and degradation of DMT1, resulting in reduced metal entry. Induction of Ndfip1 expression protects neurons from metal toxicity, and removal of Ndfip1 by shRNAi results in hypersensitivity to metals. We identify Nedd4–2 as an E3 ligase recruited by Ndfip1 for the ubiquitination of DMT1 within human neurons. Comparison of brains from Ndfip1 −/− with Ndfip1 +/+ mice exposed to Fe reveals that Ndfip1 −/− brains accumulate Fe within neurons. Together, this evidence suggests a critical role for Ndfip1 in regulating metal transport in human neurons.

Iron misregulation is a central component in the neuropathology of Parkinson's disease. The iron transport protein DMT1 is known to be increased in Parkinson's brains linking functional transport mechanisms with iron accumulation. The regulation of DMT1 is therefore critical to the management of iron uptake in the disease setting. We previously identified post-translational control of DMT1 levels through a ubiquitin-mediated pathway led by Ndfip1, an adaptor for Nedd4 family of E3 ligases. Here we show that loss of Ndfip1 from mouse dopaminergic neurons resulted in misregulation of DMT1 levels and increased susceptibility to iron induced death. We report that in human Parkinson's brains increased iron concentrations in the substantia nigra are associated with upregulated levels of Ndfip1 in dopaminergic neurons containing α-synuclein deposits. Additionally, Ndfip1 was also found to be misexpressed in astrocytes, a cell type normally devoid of this protein. We suggest that in Parkinson's disease, increased iron levels are associated with increased Ndfip1 expression for the regulation of DMT1, including abnormal Ndfip1 activation in non-neuronal cell types such as astrocytes.