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Understanding the endosomal network in neurodegeneration

Online event

Overview

Online discussion meeting organised by Professor Peter Cullen, Professor Peter St George-Hyslop, Professor Scott Small and Dr Henne Holstege.

Cryo-ET image of the yeast retromer complex associated with membrane tubules. Credit Natalya Leneva, Oleksiy Kovtun, John Briggs and Brett M Collins

Abnormalities in the endosomal network are a signature feature of Alzheimer's disease, Parkinson's disease and numerous other neurological disorders. In bringing together world leading researchers from a range of disciplines, this meeting seeks to discuss the latest advances in this rapidly moving field and foster collaborative efforts to enhance the rate of discovery in this medically important research field.

The schedule of talks and speaker biographies are available below. Speaker abstracts are also available below. 

Attending this event

This event is intended for researchers in relevant fields and will take place online. Please register to attend to receive the joining instructions closer to the meeting date.

Enquiries: contact the Scientific Programmes team

Schedule of talks

09 November

Session 1 10:00-12:05

Mechanistic endosomal cargo sorting

8 talks Show detail Hide detail

10:00-10:05 Introduction

10:05-10:25 The role of AP-5/SPG15/SPG11 in endo-lysosomal homeostasis

Dr Jennifer Hirst, Cambridge Institute for Medical Research, UK

Abstract

Adaptor protein (AP) complexes are a family of evolutionarily ancient heterotetrameric complexes, which facilitate the transport of transmembrane cargo between membranes. AP-5 is the most recently discovered AP complex, and there is limited mechanistic understanding as to how and where it functions. AP-5 is stably associated with two additional proteins that are encoded by the SPG11 and SPG15 genes, and this association is essential for the stability and membrane association of the complex. Mutations in AP5Z1 (aka SPG48), SPG11 or SPG15 cause hereditary spastic paraplegia, pointing to an overall role of the AP-5 complex in neuronal maintenance and/or function. At the cellular level, electron microscopy of SPG48, SPG11 and SPG15 patient cell lines have revealed the accumulation of aberrant endolysosomes, pointing to a role for the AP-5 complex in the homeostasis of endosomes/lysosomes. The extreme low abundance of the AP-5 complex has hampered efforts to understand its localisation and membrane recruitment. The discovery that cells starved of nutrients enhance the recruitment of AP-5 to late endosomes/ lysosomes was a key finding that allowed a more thorough and systematic approach to understanding the mechanism. The recruitment of the AP-5 complex to membrane requires the coincidence detection of both a lipid (phosphatidylinositol 3-phosphate) and protein component (Rag GTPases), providing a rationale for the dynamic association of the AP-5 complex to late endosomes/lysosomes but also uncovering a link between the AP-5 complex and the mTORC1 nutrient-sensing pathway.

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10:25-10:45 Opposing functions for retromer and Rab11 in extracellular vesicle cargo traffic at presynaptic terminals

Professor Avital Rodal, Brandeis University, USA

Abstract

Neuronal extracellular vesicles (EVs) play critical roles in intercellular communication and in propagation of pathogenic proteins in neurological disease. However, little is known about how and where cargoes are selectively packaged into neuronal EVs. Here, we found that loss of the endosomal retromer complex leads to accumulation of the EV cargoes Amyloid Precursor Protein (APP) and Synaptotagmin-4 (Syt4) at presynaptic nerve terminals of Drosophila motor neurons, and increased release of these cargoes in EVs. Conversely, EV cargo levels are reduced in rab11 mutants, and EV cargo sorting depends on a balance between Rab11-mediated loading and retromer-dependent removal from EV precursor compartments. By systematically exploring known retromer-dependent trafficking mechanisms, we found that EV cargo regulation depends on the ESCPE-1 complex, and is separable from several other previously identified roles of neuronal retromer. Rab11 and retromer have previously been implicated in competing pathways in Alzheimer’s Disease, suggesting that they could serve as therapeutic targets for limiting accumulation and release of toxic EV cargoes.

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10:45-11:05 Professor Peter Cullen, University of Bristol, UK

11:05-11:25 Endosomal coat assembly and regulation in neurodegeneration

Dr Lauren Parker Jackson, Vanderbilt University, USA

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11:25-11:45 De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

Professor Brett Collins, The University of Queensland, Australia

Abstract

The Retromer complex and associated sorting nexin (SNX) proteins are central mediators of endosomal membrane trafficking and signalling. In addition, mutations in Retromer can cause late-onset Parkinson’s disease, and Retromer can be hijacked by viral and bacterial pathogens during cellular infection. Seeking tools to modulate and probe Retromer function a series of de novo macrocyclic peptides have been discovered that bind to the trimeric Vps35-Vps26-Vps29 Retromer complex with high affinity and specificity. The majority of these cyclic peptides bind to a conserved surface on the Vps29 subunit. Crystal structures show that these peptides via use a di-peptide Pro-Leu sequence that structurally mimics other known interacting proteins including TBC1D5, VARP, and the bacterial effector RidL, and potently inhibit their association with Retromer in vitro and in cells. A unique macrocyclic peptide was found to bind Retromer at the interface between Vps35 and Vps26 subunits and can act as a potent molecular chaperone that stabilises the complex against thermal denaturation and disassembly. Importantly, binding of this peptide to Retromer does not perturb Retromer’s interaction with other essential regulatory proteins, and in fact allosterically enhances Retromer’s affinity for some proteins such as SNX27 and TBC1D5. Finally, its shown that the peptides can be used to probe the localisation and the proteome of Retromer interactions in cells. These macrocyclic peptides provide a potential toolbox for the study of Retromer and suggest new avenues for stabilising Retromer in future therapeutic approaches. 

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11:45-11:55 Selected short presentation

11:55-12:05 Selected short presentation

Session 2 13:00-15:00

Live panel discussion

1 talk Show detail Hide detail

13:00-15:00 Live panel discussion

Dr Jennifer Hirst, Cambridge Institute for Medical Research, UK
Dr Lauren Parker Jackson, Vanderbilt University, USA
Professor Brett Collins, The University of Queensland, Australia

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Session 3 16:00-17:00

Early career 'table' discussions

1 talk Show detail Hide detail

16:00-17:00 Early career 'table' discussions

Dr Jennifer Hirst, Cambridge Institute for Medical Research, UK
Dr Lauren Parker Jackson, Vanderbilt University, USA

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10 November

Session 1 10:00-12:00

Endo-lysosomal deregulation in neurodegeneration I

7 talks Show detail Hide detail

10:00-10:20 Professor Scott Small, Columbia University, USA

10:20-10:40 Roles of the endo-lysosomal system in dementia initiation and pathogenesis

Professor Rick Livesey, UCL/Great Ormond Street Institute of Child Health, UK

Abstract

Endolysosome dysfunction is emerging as an important pathogenic process in many neurodegenerative diseases, including Alzheimer’s disease, frontotemporal dementia, and lysosomal storage disorders. Using human stem cell-derived neurons, the group has found that autosomal dominant mutations in PSEN1, APP and SORL1 causal for Alzheimer’s disease all cause defects in human neuronal endolysosome function, compromising the degradative phase of autophagy. Furthermore, the proteins encoded by all three genes are localised to the endolysosomal network and act in a single pathway to regulate endolysosome function. Endolysosome dysfunction occurs in young neurons, prior to any protein aggregation, affecting endosome size and number, lysosome transport to the neuronal cell body, lysosome maturation (reflected in protease activation) and lysosome function in the degradative phase of autophagy, indicating that endolysosome dysfunction is a primary pathogenic effect of monogenic AD mutations in these genes. Tauopathies, including AD, share a feature of apparent spatial spreading through neural networks, from affected to healthy neurons. Whole genome CRISPR screens in human neurons were used to identify genes and pathways required for neuronal uptake of both monomeric and aggregated tau. In addition to key surface receptors and receptor-mediated endocytosis, those screens found that disruption of different aspects of intracellular vesicular trafficking alters tau uptake, pointing to roles for the endolysosomal system in both neurodegenerative disease initiation and progression.

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10:40-11:00 Dynein promotes retrograde transport of late endosomes in dendrites and is required for degradation of somatodendritic cargos

Dr Bettina Winckler, University of Virginia Medical School, USA

Abstract

Unlike axons, dendrites in vertebrate neurons have microtubule arrays with mixed polarities. The question of how directional transport is organised in dendrites is thus a longstanding one. Dynein has been reported as either an anterograde or retrograde motor in dendrites. The group has studied endosomal transport in dendrites, in particular the trafficking of the neuronal dendritic membrane proteins NSG1/2. They previously found that NSG1/2 was endocytosed into dendritic early endosomes and then rapidly transported via late endosomes to the soma where most of the lysosomes reside. When Rab7 function was impaired by expression of Rab7-T22N, late endosomes carrying endocytosed NSG1/2 stopped moving and accumulated high levels of NSG1/2 throughout dendrites. The group now asks if movement of late endosomes towards the soma requires kinesin or dynein motors. Since the retrograde transport and subsequent degradation of NSG1/2 depended on Rab7, they first expressed two Rab7 effectors, FYCO1 which recruits kinesin and moves lysosomes to the cell periphery in fibroblasts, and RILP which recruits dynein and moves lysosomes to the cell centre in fibroblasts. RILP overexpression leads to massive re-localisation of late endosomes to the soma whereas FYCO1 overexpression does not. This observation suggests that dynein promotes retrograde movement of late endosomes in dendrites. In order to test this hypothesis more directly, the group carried out live imaging of Rab7-mCherry. Overexpression of DIC2-GFP leads to an increase in net retrograde movements of Rab7-positive compartments, but anterograde discursions are frequent as well. In addition, they live imaged Rab7-mCherry in the presence of dynein inhibitors (CC1-GFP, ciliobrevin). Both of these approaches largely halt Rab7 movements in dendrites. The group then determined if degradation of NSG1/2 was slowed if dynein function is disrupted with CC1 overexpression. They find that NSG1/2 accumulates in dispersed compartments in dendrites and degradation is slowed. In addition, they find reduced accumulation of acidified (Lysotracker-positive) compartments in somata of CC1-GFP expressing neurons. Lastly, DQ-BSA conversion to red (a degradative tracer) is reduced in the somata of CC1-GFP expressing neurons. These data show that late endosomes use dynein motors for net motility to the soma and thus use dendritic plus end-out microtubules as their tracks for motility.

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11:00-11:20 Speaker tbc

11:20-11:40 Endosomal trafficking is required for the normal maturation of the Alzheimer’s-associated protein SORLA

Professor Olav Andersen, Aarhus University, Denmark

Abstract

SORL1 is among the most significant genetic factors that affect development of Alzheimer’s disease (AD). This gene encodes a sorting receptor, SORLA, involved in trafficking of multiple different cargoes between cell surface and golgi/endosomal compartments.

SORLA undegoes posttranslational modifications and maturation with ultimate ectodomain shedding, however knowledge of these processes remains limited. Here it is demonstrated that SORLA exists in two forms at the plasma membrane, an immature and a mature form, characterised by distinct N-glycosylation profiles. The mature receptor form is shed from the cell surface, whereas immature form of sorLA does not undergo shedding. Conversion of the immature to the mature N-glycan profile relies on endocytosis and recycling of the receptor to the cell surface by a retromer-dependent endosomal trafficking pathway.

Understanding the maturation process of SORLA has implications for assessment of genetic SORL1 variants identified in AD patients, as mutations that cause misfolding or trafficking dysfunction are likely to impair maturation. Therefore, the data presented point to impaired maturation as a signature of AD-associated SORLA dysfunction. This can be utilised for future functional studies to assess pathogenicity of genetic SORL1 variants.

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11:40-11:50 Selected short presentation

11:50-12:00 Selected short presentation

Session 2 13:00-15:00

Live panel discussion

1 talk Show detail Hide detail

13:00-15:00 Live panel discussion

Professor Rick Livesey, UCL/Great Ormond Street Institute of Child Health, UK
Dr Bettina Winckler, University of Virginia Medical School, USA
Professor Olav Andersen, Aarhus University, Denmark

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Session 3 16:00-17:00

Early career 'table' discussions

1 talk Show detail Hide detail

16:00-17:00 Early career 'table' discussions

Professor Rick Livesey, UCL/Great Ormond Street Institute of Child Health, UK
Dr Bettina Winckler, University of Virginia Medical School, USA
Professor Olav Andersen, Aarhus University, Denmark

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11 November

Session 1 10:00-12:00

Endo-lysosomal deregulation in neurodegeneration II

7 talks Show detail Hide detail

10:00-10:20 Deciphering the role of the LRRK2-Rab signalling pathway in Parkinson’s disease

Professor Dario Alessi, University of Dundee, UK

Abstract

Autosomal dominant missense mutations that hyperactivate the LRRK2 protein kinase are a common cause of inherited Parkinson’s disease and therapeutic efficacy of LRRK2 inhibitors is being tested in clinical trials. Alessi will give an overview of the nuts and bolts of current research that has revealed that LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with a new set of effectors such as RILPL1/2 and JIP3/JIP4. He will discuss what is known about how LRRK2 regulates ciliogenesis, the endosomal-lysosome system, and immune responses and how this might be linked to Parkinson’s disease. He will consider how mutations in other components linked to Parkinson’s such as  Rab29 and VPS35 promote LRRK2 mediated Rab protein phosphorylation. He will discuss the identification and characterisation of a novel PPM family phosphatase member that counteracts LRRK2 signalling by dephosphorylating Rab proteins. Alessi will present data that indicates that the LRRK2 parlog termed LRRK1 also functions as a Rab kinase.  

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10:20-10:40 Unravelling the downstream effects of endolysosomal dysfunction on the gut-brain axis in Parkinson's disease

Dr Kerri Kinghorn, University College London, UK

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10:40-11:00 Activation of the Parkinson’s associated kinase LRRK2 at lysosomal membranes

Dr Mark Cookson, NIA, NIH, USA

Abstract

Gain of function mutations in the LRRK2 gene that encodes Leucine-rich repeat kinase 2 cause autosomal dominant Parkinson’s disease (PD). Additionally, non-coding variation at the LRRK2 locus contributes to genetic risk of sporadic PD. Prior data has strongly implicated LRRK2 in intracellular membrane trafficking by virtue of localisation to multiple cellular membranes and by phosphorylation of membrane associated Rab proteins. Furthermore, a prominent feature of LRRK2 knockout mice is the accumulation of swollen and enlarged lysosomes, suggesting that the endogenous LRRK2 protein regulates some aspect of lysosomal trafficking. The group has recently shown that lysosomal damage induced by exposure of cultured cells to L-leucyl-L-leucine methyl ester (LLOMe) causes recruitment of LRRK2 to the lysosomal membrane and activation of kinase activity. This results in the phosphorylation of lysosomal Rab proteins that then interact with the cytoskeletal adaptor protein JIP4 and leads to tubulation of the lysosomal membrane to form vesicles that can migrate to undamaged lysosomes in the same cell. The group has named this process LYTL (lysosomal tubulation induced by LRRK2) and further show that (1) coding mutant forms of LRRK2 enhance LYTL leading to excessive tubulation in cultured astrocytes and that (2) non-coding variation in the 5’ region of the LRRK2 gene also enhances LRRK2 activation in response to LLOMe in IPSC-derived microglial cells. These results imply that multiple types of genetic variation around LRRK2 share the same direction of effect and, by extension, suggest that inhibiting LRRK2 may be a novel therapeutic approach for both inherited and sporadic PD.

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11:00-11:20 Endo-lysosomal dysfunction – a unifying mechanism for hereditary spastic paraplegia

Dr Evan Reid, Cambridge Institute of Medical Research, UK

Abstract

Hereditary spastic paraplegias are genetic conditions in which corticospinal tract axons selectively generate. Mutations in the microtubule severing ATPase spastin are the most common cause of the condition. In this talk Dr Reid will describe his work in studying the role of spastin at endosomes and how abnormality of this translates to lysosomal dysfunction in axons. He will demonstrate that spastin is required for efficient endosomal tubule fission at the point of contacts between endosomal tubules and the endoplasmic reticulum. Defective endosomal tubule fission in cells lacking spastin has consequences for receptor sorting away from the endosomal pathway; notably it causes a block in endosome-to-Golgi mannose 6-phosphate receptor (M6PR) traffic. The normal function of M6PR is to collect lysosomal enzymes from the Golgi and traffic them back to the endolysosomal compartment, so lack of M6PR at the Golgi in cells lacking spastin is associated with abnormal lysosomal enzyme traffic and defects in lysosomal morphology and function. In neurons from a spastin-HSP knock-in mouse model and in human neurons derived reprogrammed from spastin patient fibroblasts, abnormal lysosomes are found within pathological axonal swellings, potentially explaining the axonopathy in spastin-HSP. Work will also be presented that indicates that lysosomal dysfunction is a unifying pathological mechanism across many different HSP genetic subtypes. 

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11:20-11:40 Hyperactive LRRK2 kinase alters neuronal autophagy by disrupting the axonal transport of autophagosomes

Professor Erika Holzbaur, University of Pennsylvania, USA

Abstract

Neurons rely on autophagy, a critical homeostatic mechanism, to maintain cellular health over the decades of human life. Deficits in autophagy cause the accumulation of protein aggregates and dysfunctional mitochondria, and are characteristic of major neurodegenerative diseases including Parkinson’s disease (PD). The group has identified a constitutive and nonselective pathway for axonal autophagy, in which autophagosomes are generated preferentially in the distal axon and then rapidly transported to the soma by molecular motors interacting with the microtubule cytoskeleton. Here, the group asked how this pathway is altered in neurons expressing the most common PD-causing mutation in LRRK2; the G2019S mutation induces hyperactive LRRK2 kinase activity leading to increased phosphorylation of Rab GTPases that regulate intracellular trafficking. They found that LRRK2-G2019S significantly reduces the processivity of autophagosome transport along axons. Similar effects were observed across multiple models, including hippocampal neurons overexpressing mutant LRRK2, cortical neurons from a knock-in mouse, and human iPSC-derived neurons gene-edited to express the G2019S mutation. Altered transport led to impaired organelle maturation; this deficit was reversed by pharmacological inhibition of LRRK2 kinase activity. The group probed the underlying mechanism, and found that hyperactive LRRK2 recruits the motor adaptor JIP4 to the autophagosomal membrane, inducing abnormal activation of kinesin and resulting in a tug-of-war between anterograde and retrograde motors bound to the organelle and thus inhibition of processive unidirectional motility. Together, these findings demonstrate that increased LRRK2 kinase activity is sufficient to induce defects in autophagosome transport and maturation, further implicating defective autophagy in the pathogenesis of Parkinson’s disease.  

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11:40-11:50 Selected short presentation

11:50-12:00 Selected short presentation

Session 2 13:00-15:00

Live panel discussion

1 talk Show detail Hide detail

13:00-15:00 Live panel discussion

Professor Dario Alessi, University of Dundee, UK
Dr Kerri Kinghorn, University College London, UK
Dr Evan Reid, Cambridge Institute of Medical Research, UK

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Session 3 16:00-17:00

Early career 'table' discussions

1 talk Show detail Hide detail

16:00-17:00 Early career 'table' discussions

Professor Dario Alessi, University of Dundee, UK
Dr Kerri Kinghorn, University College London, UK
Dr Evan Reid, Cambridge Institute of Medical Research, UK

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12 November

Session 1 10:00-12:00

Genetics of endo-lysosomal neurodegenerative disease

7 talks Show detail Hide detail

10:00-10:20 Dr Henne Holstege, Amsterdam UMC, The Netherlands

10:20-10:40 Professor Peter St George-Hyslop FRS, Cambridge Institute for Medical Research, UK and University of Toronto, Canada

Professor Peter St George-Hyslop FRS, Cambridge Institute for Medical Research, UK and University of Toronto, Canada

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10:40-11:00 Rescuing autophagosome and lysosome defects in C9ORF72 ALS/FTD by inhibiting PIKFYVE kinase

Dr Justin Ichida, University of Southern California, USA

Abstract

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), the group found that repeat-expanded C9ORF72 was haploinsufficient in ALS. They found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels, augmenting its function with constitutively active RAB5, or compensating for its loss by inhibiting PIKFYVE and activating secretory autophagy ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, these results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

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11:00-11:20 Therapeutic effects of retromer stabilization in Amyotrophic Lateral Sclerosis models

Professor Pierfausto Seneci, University of Milan, Italy
Dr Luca Muzio, San Raffaele Scientific Institute, Italy

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterised by the degeneration of upper and lower motor neurons (MNs). Protein aggregation and misfolding likely contribute to MNs cell death, and therefore their clearance may represent a therapeutic strategy with the potential to enter the clinical practice. The retromer is a multi-protein complex involved in retrograde transport of proteins from endosomes to the trans-Golgi network and to the cell membrane. The group found significantly reduced levels of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases – a pathological hallmark in ALS – they rationally designed and synthesized an array of retromer stabilisers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. Such molecules, working as a pharmacological chaperone, increased the stability/functioning of retromer in vitro and in vivo. Among them, the group selected leading compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuated locomotion impairment and increased MNs survival. Moreover, compound 2a increased VPS35 in iPSCs-derived MNs and showed a high brain bioavailability. These results clearly suggest the retromer as a valuable 'druggable' target in ALS.

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11:20-11:40 Speaker tbc

11:40-11:50 Selected short presentation

11:50-12:00 Selected short presentation

Session 2 13:00-15:00

Live panel discussion

Session 3 16:00-17:00

Early career 'table' discussions

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