Centre for Neuroscience - Theses
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ItemThe control of Nedd4 family interacting protein 1 (Ndfip1) expression and its binding partners( 2012)The Nedd4 family interacting protein 1 (Ndfip1) is a neuroprotective protein, highly up-regulated in the neuron following brain injury. Many fundamental questions regarding the functions and regulations of Ndfip1 remained unsolved. Therefore, this study aimed to investigate Ndfip1 from three different aspects: 1) novel drugs or compounds which up-regulate the Ndfip1 levels, 2) novel binding partners of Ndfip1 and 3) specific functions and downstream processes of following the interaction between Ndfip1 and PTEN. A high-throughput screening method was developed in this study in order to search the novel drugs or compounds which up-regulate Ndfip1 level. Funneling from the large collection of bioactive compounds (4400 compounds), this process had short-listed 23 compounds which potentially up-regulate the levels of Ndfip1. Six compounds from the short-listed potential candidates were validated and successfully up-regulate Ndfip1 levels in various cell lines. The search of novel Ndfip1 binding partners involved with a two-step purification technique, known as tandem affinity purification. By using Ndfip1 as a bait protein, a large network of protein candidates binding with Ndfip1 was identified. Among these proteins candidates, the FANCD2 (Fanconi anemia group D2 protein) was validated using Duolink technique, and its interaction with Ndfip1 was confirmed. FANCD2 is a pivotal protein involved in DNA damage signaling and repair. The association between Ndfip1 and FANCD2 suggest that Ndfip1 might play a role in regulating the DNA damage signaling and repair. The most prominent impact derived from this study is the discovery of Ndfip1 and PTEN interaction. PTEN is a tumor suppressor protein which involved in the tumor formation if PTEN is mis-regulated. In recent years, more reports associated PTEN with the role of neuroprotection. This interaction between Ndfip1 and PTEN enhanced PTEN ubiquitination. Ndfip1 mediated PTEN ubiquitination resulting in PTEN nuclear translocation, but not degradation. Furthermore, this Ndfip1-PTEN interaction is dominantly regulated by PTEN phosphorylation. The contributions of this study include gaining better understanding regarding the control of Ndfip1 levels, as well as the network of proteins which bind to Ndfip1. This study also provided a mechanistic understanding regarding the neuroprotective function of Ndfip1. Here, I would like to propose that the increased level of Ndfip1 during neuronal injury, leads to PTEN nuclear translocation, and promote cell survival through the regulation of p-Akt levels in stressed neurons. Therefore, it will be in great interest to follow-up the leads from this study, in order to understand in greater detail the specific neuroprotective mechanism of Ndfip1.
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ItemMolecular mechanisms of Ndfip1 during brain development and consequences for neuronal survival( 2012)In 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.