Retromer-dependent traffic and tau secretion: mechanistic and therapeutic implications
Professor Scott Small, Columbia University, USA
Retromer is the dominant trafficking complex that recycles cargo out of endosomes. Retromer was first linked to Alzheimer’s disease by expression profiling studies that set out to understand why the entorhinal cortex is differentially vulnerable to the disease. Since then many studies have established a mechanistic link to Alzheimer’s disease. By using manipulations that either decrease or increase retromer recycling in model systems, retromer has been shown to mediate the disease’s four core pathologies: Amyloid pathology, tau pathology, synaptic pathology, and microglia pathology. While retromer recycling defects exist in, and are pathogenically linked, to the disease, the frequency of retromer dysfunction remains unknown. To address this question, the group set out to identify a biomarker of retromer dysfunction in the cerebrospinal fluid (CSF). They began by genetically-engineering a mouse to have retromer dysfunction selectively in forebrain neurons. A proteomic screen of CSF and subsequent validation studies showed that retromer dysfunction is associated with CSF elevations of APLP1, CHL1, and the microtubule binding protein tau. Next, the group turned to human studies, and developed new assays that allow them to reliably measure all three proteins in human CSF. By establishing the relationship of all three proteins in the CSF of healthy controls and Alzheimer patients in the dementia and prodromal stages disease, the results suggest that retromer recycling dysfunction might exist in a majority of patients. These findings have both mechanistic and therapeutic implications.
Roles of the endo-lysosomal system in dementia initiation and pathogenesis
Professor Rick Livesey, UCL/Great Ormond Street Institute of Child Health, UK
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.
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
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.
The impact of SORL1 loss on endo-lysosomal network function in human CNS cells
Dr Jessica Young, University of Washington, USA
Enlarged early endosomes, indicative of endosomal traffic jams, are a hallmark cytopathology in Alzheimer’s Disease. The sorting receptor SorLA, encoded by the established risk gene SORL1, has a defined role in endolysosomal trafficking of the amyloid precursor protein, APP. Research in the Young lab has demonstrated that loss of SORL1 in hiPSC-derived neurons leads to enlarged early endosomes in an amyloid-independent manner and that this endosome enlargement can be rescued by treatment with retromer-stabilizing molecules. Further work in the lab shows that modulation of SORL1 expression affects the sorting of neuronal cargo other than APP, and functionally demonstrates a role for SORL1 in lysosomal trafficking in both hiPSC-derived neurons and microglia. RNA-seq analysis of SORL1 deficient neurons indicates altered expression and network interaction changes in neurotrophin and synaptic genes, demonstrating that endosomal traffic jams induced by loss of SORL1 impair pathways necessary for neuronal health and function. Collectively, these studies suggest that SORL1 plays multiple roles in central nervous system cells and that strategies enhancing endosomal trafficking should be considered as therapeutic targets for AD.
Endosomal trafficking is required for the normal maturation of the Alzheimer’s-associated protein SORLA
Professor Olav Andersen, Aarhus University, Denmark
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.
Selected short presentation
Regulation of lipid trafficking at endoplasmic reticulum-plasma membrane contact sites in photoreceptor neurons
Bishal Basak, Harini Krishnan and Padinjat Raghu
National Centre for Biological Sciences, TIFR-GKVK Campus, India
At the distal regions of neurons, vesicular trafficking is inefficient to maintain lipid homeostasis. Instead, the cell relies on contacts formed between closely apposed organellar membranes for lipid transfer. Perturbations of contacts formed between the endoplasmic reticulum [ER] and mitochondria have been implicated in neurodegeneration. However, the mechanisms by which these contact sites directly contribute to maintaining neuronal physiology remain unclear.
Drosophila photoreceptors are morphologically polarized neurons forming contacts between the apical plasma membrane [PM] and ER, which are indispensable for maintaining cellular integrity. RDGB is a key protein that tightly controls this process by transferring phospholipids at the contact sites, which in turn is essential to maintain organellar lipid homeostasis. Electrophysiological recordings from photoreceptors devoid of RDGB function show significant reduction in light response and sensitivity, subsequently leading to retinal degeneration. This study demonstrates that the function of RDGB at the ER-PM junction depends on interaction with phospholipids at the PM and also with the integral ER protein, VAP. Additionally, inter-domain movements within the RDGB protein directly regulate its lipid transfer function. Disruption of these interactions severely impacts lipid homeostasis leading to photoreceptor degeneration. Thus, this study provides insight into how regulation of lipid transfer is fine-tuned to maintaining neuronal structure and function.
Selected short presentation
TMCC2, an endosomal/ER protein that differentially interacts with isoforms of apolipoprotein E and the Amyloid Precursor Protein associated with Alzheimer’s Disease
Paul CR Hopkins, Michelle Lupton, Richard Killick, John Hardy, John Powell and Guy Tear
Centre for Developmental Neurobiology, King’s College London, UK
Transmembrane and Coiled-Coil 2 (TMCC2) is a protein that regulates endosome fission at the ER, and forms complexes with both apolipoprotein E (apoE) and the Amyloid Precursor Protein (APP). TMCC2 demonstrates isoform-specific interactions, interacting differentially with risk versus normal forms of both proteins. TMCC2 is a member of a highly evolutionarily conserved gene family; the Drosophila orthologue (called Dementin) is essential for brain development, modifies metabolism of the APP-Like protein in vivo and protects against mis-expression of human APP. Dementin alleles cause neurodegeneration with features of Alzheimer’s disease (AD), ie accumulation of fragments of the APP-Like protein, defects in synaptogenesis, cellular mis-localization of microtubule-binding proteins and early death.
The group performed targeted exome sequencing of TMCC2 in AD cases and controls and found an excess of TMCC2 variants in AD cases. Several of these had modified activity towards apoE and APP that affected the amount of Aβ secreted from cultured human cells. TMCC2 is abundantly expressed in the human brain where anti-TMCC2 antibodies detect a primarily neuronal expression pattern.
TMCC2 may thus act to link apoE isoforms and APP to defects of the endo-lysosomal system in AD.