Periodic Reporting for period 1 - SeCRT (Uncover mechanisms of unconventional secretion of tau using functional CRISPR screens - From basic discoveries to neurodegenerative disease therapeutics)
Período documentado: 2019-04-01 hasta 2021-03-31
Most of secreted proteins transit through the endoplasmic reticulum (ER) and the Golgi apparatus. However, eukaryotic cells secrete cytosolic proteins, which do not enter the ER. Proteins that use this “unconventional secretory pathway” include factors with critical functions and deregulation of their transport is associated to a wide range of diseases. Yet, mechanisms of unconventional secretion remain largely uncharacterised. This represents a major gap in our understanding of fundamental mechanisms supporting protein trafficking and secretion. During my previous study, I developed a powerful platform for pooled genome-wide CRISPRi screening with the aim of identifying genes involved in protein transport in mammalian cells. In this proposal, I will use this approach to uncover mechanisms involved in the unconventional secretion of tau. During aging, abnormal accumulation of tau is a common pathological hallmark of neurodegenerative diseases (ND), including Alzheimer’s disease, for which there are no efficient therapeutic to prevent/slow down the pathogenesis. Compelling evidences suggest that tau aggregation occur via its transmission from neuron to neuron. Thus, tau secretion appears to be a pivotal event in promoting ND progression. For these reasons, to understand how tau is secreted is of critical importance. Objectives that will be addressed in this project will be to identify new factors required for tau secretion using a pooled genome-wide CRISPRi screen. Then, by combining biochemical and cellular approaches, as well as the last technologies for imaging, candidate genes will be functionally characterized. Finally, the most relevant factors will be evaluated as potential therapeutic target in zebrafish model of ND. This project will allow uncovering fundamental mechanisms to understand ND progression associated to the unconventional secretion of tau and could ultimately lead to the discovery of effective strategies to develop new therapies.
Main achievements:
I characterised experimental conditions associated to the unconventional secretion of Tau. Using the SH-SY5Y cell line and mouse primary neurons, I established that their culture in a normal growth medium enables Tau detection in cell supernatants, while culture under starvation conditions is associated to cell toxicity. Endogenous Tau expression was assessed as well as ectopic expression in cells expressing Tau in a transient or stable manner. Comparative analysis of Tau variants has also been initiated and results do not show major differences of Tau secretion.
I developed new assays to monitor unconventional secretion. Constructs required to engineer cell lines used for the proposed assays have been generated, and their expression tested. Constructs have been generated for monitoring Tau secretion in comparison with other unconventionally secreted proteins (α-synuclein and Galectin-3). The same reporter constructs will be employed for both assays whereas distinct hook constructs will be used. For the trapping assay at the cell surface, a construct allowing the expression of CD8 fused to St has been generated and its expression tested. The retention of Tau at the cell surface has been assessed by immunofluorescence and flow cytometry. For the assay based on the split luciferase detection, I assessed the presence of reporters in the supernantant and at the cell surface. This assay is sensitive, with a signal to noise ratio above 2-log. Clonal stable cell lines have been generated with these constructs and will be used to develop pooled genome-wide CRISPR screen and drug screen.
I started a targeted approach to characterize mechanisms involved in Tau secretion, based on the specific knockdown of particular genes. Preliminary data suggest that secretory lysosomes are required for the unconventional secretion of Tau.
I characterised experimental conditions associated to the unconventional secretion of Tau. Using the SH-SY5Y cell line and mouse primary neurons, I established that their culture in a normal growth medium enables Tau detection in cell supernatants, while culture under starvation conditions is associated to cell toxicity. Endogenous Tau expression was assessed as well as ectopic expression in cells expressing Tau in a transient or stable manner. Comparative analysis of Tau variants has also been initiated and results do not show major differences of Tau secretion.
I developed new assays to monitor unconventional secretion. Constructs required to engineer cell lines used for the proposed assays have been generated, and their expression tested. Constructs have been generated for monitoring Tau secretion in comparison with other unconventionally secreted proteins (α-synuclein and Galectin-3). The same reporter constructs will be employed for both assays whereas distinct hook constructs will be used. For the trapping assay at the cell surface, a construct allowing the expression of CD8 fused to St has been generated and its expression tested. The retention of Tau at the cell surface has been assessed by immunofluorescence and flow cytometry. For the assay based on the split luciferase detection, I assessed the presence of reporters in the supernantant and at the cell surface. This assay is sensitive, with a signal to noise ratio above 2-log. Clonal stable cell lines have been generated with these constructs and will be used to develop pooled genome-wide CRISPR screen and drug screen.
I started a targeted approach to characterize mechanisms involved in Tau secretion, based on the specific knockdown of particular genes. Preliminary data suggest that secretory lysosomes are required for the unconventional secretion of Tau.
I am developing advanced tools that will open stimulating perspectives in the field of unconventional secretion. I will use these assays to decipher the molecular mechanisms underlying Tau secretion. Targeted approaches will allow to discriminate whether Tau is transported from the cytosol to the extracellular space by direct translocation or through membrane-bound compartments that can derive from autophagic membranes, endosomes and MVBs or secretory lysosomes. Then critical factors at every step of Tau transport should be highlighted thanks to the pooled genome-wide CRISPRi screen. If vesicular compartments are involved, I will reveal how Tau is packed into transport carriers by identifying specific chaperone(s) and transporter(s). I also expect to uncover the source of the membrane for the formation of these transport carriers and if different vesicular intermediates are involved. Identification of the processes of biogenesis, transport and fusion with the membrane of these vesicular intermediates will be of particular interest to characterize the dynamics of these events.
Finally, I expect to identify factors that could be used in the longer-term as potential therapeutic targets for neurodegenerative diseases (NDs), by using Zebrafish models. It will provide a unique opportunity to pinpoint specific genes involved in Tau secretion and the downregulation of which would prevent ND progression.
Taken together, this research program at the crossroad of critical challenges can make important advances into basic cell biology principles. In addition, it is likely that results obtained will yield novel mechanistic insights that would be exploited for biomedical applications. Hence, this project should also have an important impact in the medical, social and economic fields.
Medical applications: The cause of NDs such as AD remains poorly understood, and there are currently no medications to stop or reverse its progression. For this reason, there is an urgent need for innovative treatments and for early biomarkers of the disease. Unravelling mechanisms involved in the trans-cellular spreading of pathological protein species will pave the way for new strategies for its treatment.
Social and economic applications: The incidence of NDs such as Alzheimer’s disease is growing globally, becoming one of the greatest scourges to human health. In 2015, there were approximately 29.8 million people worldwide with Alzheimer’s disease, generating high economical and societal burdens. In addition, currently available treatments are only symptomatic and they are inevitably associated during the progression of the disease to psychosocial and caregiving treatments. Thus, Alzheimer’s disease is among the most financially costly diseases for society in Europe and US. Therefore, any treatment that slows cognitive decline, delays institutionalization or reduces caregivers’ hours will have economic benefits.
Finally, I expect to identify factors that could be used in the longer-term as potential therapeutic targets for neurodegenerative diseases (NDs), by using Zebrafish models. It will provide a unique opportunity to pinpoint specific genes involved in Tau secretion and the downregulation of which would prevent ND progression.
Taken together, this research program at the crossroad of critical challenges can make important advances into basic cell biology principles. In addition, it is likely that results obtained will yield novel mechanistic insights that would be exploited for biomedical applications. Hence, this project should also have an important impact in the medical, social and economic fields.
Medical applications: The cause of NDs such as AD remains poorly understood, and there are currently no medications to stop or reverse its progression. For this reason, there is an urgent need for innovative treatments and for early biomarkers of the disease. Unravelling mechanisms involved in the trans-cellular spreading of pathological protein species will pave the way for new strategies for its treatment.
Social and economic applications: The incidence of NDs such as Alzheimer’s disease is growing globally, becoming one of the greatest scourges to human health. In 2015, there were approximately 29.8 million people worldwide with Alzheimer’s disease, generating high economical and societal burdens. In addition, currently available treatments are only symptomatic and they are inevitably associated during the progression of the disease to psychosocial and caregiving treatments. Thus, Alzheimer’s disease is among the most financially costly diseases for society in Europe and US. Therefore, any treatment that slows cognitive decline, delays institutionalization or reduces caregivers’ hours will have economic benefits.