Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following 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.Jason Howitt, Jenny Lackovic, Ley-Hian Low, Adam Naguib, Alison Macintyre, Choo-Peng Goh, Jennifer K. Callaway, Vicki Hammond, Tim Thomas, Matthew Dixon, Ulrich Putz, John Silke, Perry Bartlett, Baoli Yang, Sharad Kumar, Lloyd C. Trotman, and Seong-Seng Ta

Molecular mechanisms of Ndfip1 during brain development and consequences for neuronal survival

© 2012 Dr. Choo-Peng GohIn this thesis, I describe experiments to study the function of Ndfip1 (Nedd4 family-interacting protein 1), an adaptor for Nedd4 family of ubiquitin ligases, during development of the mouse neocortex and its response following traumatic brain injury (TBI). Ndfip1 functions by binding and recruiting proteins for ubiquitination by Nedd4-family ubiquitin ligases, comprising nine members. Ubiquitinated proteins can be either degraded in the proteasome apparatus or become signaling proteins. Either way, it has a profound impact on the subsequent behaviour of neurons during development or disease. In the present study, I first investigated the spatial and temporal expression pattern of Ndfip1 during cortical development. In the early stages (embryonic day 11, E11), Ndfip1 is highly expressed in proliferative cells of the germinal zone where neurons are born. As development progresses (E15 onwards), Ndfip1 expression shifts to the mature neurons in the cortical plate, with concomitant reduction in the ventricular zone. This dynamic shift from proliferative to non-proliferative regions of the cortex suggests a dual role for Ndfip1 in proliferating neurons and mature neurons. To explore this, I investigated the relationship between Ndfip1 and Sprouty2 (Spry2), an inhibitor of cell division and cell survival via the MAP-kinase pathway. I provide evidence to demonstrate that Ndfip1 binds to Spry2, in both endogenous and over-expression systems. In the developing cortex, Ndfip1 and Spry2 expression are similar and coincidental. In a neuronal cell line (SY5Y), artificial over-expression of Ndfip1 results in reduction of Spry2, suggesting that Ndfip1 can down-regulate Spry2 levels most likely by Nedd4 ubiquitination. Consistent with this notion, the levels of Spry2 are upregulated following Ndfip1 loss in Ndfip1-/- fibroblasts. This upregulation of Spry2 is associated with attenuated epidermal growth factor-elicited ERK1/2 signaling. Therefore, association of Ndfip1 with Spry2 might be important for the regulation of of Spry2 and the MAP-kinase signaling pathway during cortical development. I have also investigated the potential role of Ndfip1 as a neuroprotective agent following brain injury by using a mouse model of closed head injury. Ndfip1 was upregulated in surviving neurons close to the trauma lesion at 6 h and 24 h post-TBI. Given that Ndfip1 can bind and mediate ubiquitination of the tumor suppressor PTEN, I investigated the relationship between the two. I demonstrate, for the first time, that Pten is translocated to the neuronal nucleus following brain injury. This event is coincident with Ndfip1 upregulation and survival of neurons situated close to the sites of lesion. I performed biochemical assays that revealed that Pten levels remained stable after TBI, suggesting that nuclear Pten in cortical neurons results from relocation of existing cytoplasmic Pten rather than Pten upregulation. In vivo, I also show that Ndfip1 upregulation and Pten nuclear trafficking are events associated with neuronal survival after TBI, as mice lacking Ndfip1 sustained larger brain lesions compared to wild-type controls. In addition Ndfip1 upregulation is correlated with increased Akt phosphorylation in the trauma hemisphere, suggesting that neuron survival is associated with higher p-Akt levels. Finally, I demonstrate that TBI induces increase binding of Ndfip1 to Nedd4-2, but not Nedd4-1 indicating that Nedd4-2 is the E3 ligase for Ndfip1 in the brain. I conclude that in brain injury, Ndfip1 together with Nedd4-2 is neuroprotective in surviving neurons by trafficking Pten into the nucleus, rather than by degrading cytoplasmic Pten. These experiments firmly establish that, through Pten, Ndfip1 is a key regulator of PI3-kinase signaling for cell survival following brain injury. In summary, the experiments in my thesis provide novel evidence that Ndfip1 is an important player during development of the cortex in the embryo, and protection of the cortex in the adult. In either scenario, Ndfip1 functions by regulating the MAP-kinase and PI3-kinase pathways, which are known to control a multitude of cellular functions including cell growth and cell survival. I provide strong evidence to suggest that Ndfip1 regulates the negative regulators (PTEN and Spry2 respectively) of PI3-kinase and MAP-kinase signaling pathways. My work offers the foundation for future strategies to manipulate Ndfip1 for improving neuron survival

Rab5 and Ndfip1 Are Involved in Pten Ubiquitination and Nuclear Trafficking

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. The subcellular distribution of Pten is critical for its multiple roles as a tumour suppressor. Here we describe the trafficking of Pten on both early and recycling endosomes using bimolecular fluorescence complementation. We show that both Rab5 GTPase and the E3 ligase adaptor protein Ndfip1 are required for ubiquitination and nuclear trafficking of Pten

Nuclear trafficking of Pten after brain injury leads to neuron survival not death

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

Clinical outcome and prognostic factors for Asian patients in Phase I clinical trials

10.1038/s41416-023-02193-2BRITISH JOURNAL OF CANCER128

Single administration of Selective Internal Radiation Therapy versus continuous treatment with sorafeNIB in locally advanced hepatocellular carcinoma (SIRveNIB): study protocol for a phase iii randomized controlled trial

BACKGROUND: Approximately 20 % of hepatocellular carcinoma (HCC) patients diagnosed in the early stages may benefit from potentially curative ablative therapies such as surgical resection, transplantation or radiofrequency ablation. For patients not eligible for such options, prognosis is poor. Sorafenib and Selective Internal Radiation Therapy (SIRT) are clinically proven treatment options in patients with unresectable HCC, and this study aims to assess overall survival following either SIRT or Sorafenib therapy for locally advanced HCC patients. METHODS: This investigator-initiated, multi-centre, open-label, randomized, controlled trial will enrol 360 patients with locally advanced HCC, as defined by Barcelona Clinic Liver Cancer stage B or stage C, without distant metastases, and which is not amenable to immediate curative treatment. Exclusion criteria include previous systemic therapy, metastatic disease, complete occlusion of the main portal vein, or a Child-Pugh score of >7. Eligible patients will be randomised 1:1 and stratified by centre and presence or absence of portal vein thrombosis to receive either a single administration of SIRT using yttrium-90 resin microspheres (SIR-Spheres®, Sirtex Medical Limited, Sydney, Australia) targeted at HCC in the liver by the trans-arterial route or continuous oral Sorafenib (Nexavar®, Bayer Pharma AG, Berlin, Germany) at a dose of 400 mg twice daily until disease progression, no further response, complete regression or unacceptable toxicity. Patients for both the Sorafenib and SIRT arms will be followed-up every 4 weeks for the first 3 months and 12 weekly thereafter. Overall survival is the primary endpoint, assessed for the intention-to-treat population. Secondary endpoints are tumour response rate, time-to-tumour progression, progression free survival, quality of life and down-staging to receive potentially curative therapy. DISCUSSION: Definitive data comparing these two therapies will help to determine clinical practice in the large group of patients with locally advanced HCC and improve outcomes for such patients.BioMed Central open acces