Daly JL, Danson CM, Lewis PA, Zhao L, Riccardo S, Di Filippo L, Cacchiarelli D, Lee D, Cross SJ, Heesom KJ, Xiong WC, Ballabio A, Edgar JR, Cullen PJ. Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health. Nat Commun. 2023 May 29;14(1):3086. PubMed.

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  1. The association of the multi-protein sorting complex, retromer, with neurodegenerative disorders, such as AD and PD, has long been known. The immense complexity of the pathway, however, makes dissecting the mechanisms of this association challenging.

    This exciting new work by the Cullen group has just made large strides in uncovering these mechanisms. By using VPS35 KO cells and performing lysosomal immunoprecipitation, they have meticulously detailed changes in proteins associated with endo-lysosomal organelles and the cell surface. The data indicate that loss of retromer could promote cell stress, leading to neurodegeneration by failure of resolution of lysosome membrane dynamics and an increase in lysosomal exocytosis. This could release potentially pathogenic proteins outside the cell where they can seed aggregation in neighboring cells.

    Surprisingly, their data also indicates a role for retromer in regulating lysosomal identity, which, when not acquired properly, can change cellular metabolic programs. Several studies have used small-molecule retromer chaperones to improve AD phenotypes (Mecozzi et al, 2014; Young et al., 2018; Mishra et al., 2022). A fuller understanding of the intricate processes regulated by retromer in the cell will certainly aid in studies aimed to therapeutically target this pathway.

    References:

    Mecozzi VJ, Berman DE, Simoes S, Vetanovetz C, Awal MR, Patel VM, Schneider RT, Petsko GA, Ringe D, Small SA. Pharmacological chaperones stabilize retromer to limit APP processing. Nat Chem Biol. 2014 Jun;10(6):443-9. Epub 2014 Apr 20 PubMed.

    Young JE, Fong LK, Frankowski H, Petsko GA, Small SA, Goldstein LS. Stabilizing the Retromer Complex in a Human Stem Cell Model of Alzheimer's Disease Reduces TAU Phosphorylation Independently of Amyloid Precursor Protein. Stem Cell Reports. 2018 Mar 13;10(3):1046-1058. Epub 2018 Mar 1 PubMed.

    Mishra S, Knupp A, Kinoshita C, Martinez R, Theofilas P, Young JE. Pharmacologic Stabilization of Retromer Rescues Endosomal Pathology Induced by Defects in the Alzheimer's gene SORL1. 2022 Aug 02 10.1101/2022.07.31.502217 (version 1) bioRxiv.

    View all comments by Jessica Young

  2. Retromer abnormalities have been implicated in Alzheimer’s disease pathogenesis, but the exact mechanisms remain unclear. This new study by Daly et al. offers valuable new insights by linking retromer with endo-lysosomal system health; specifically, the study shows how H4 neuroglioma cells knocked out for the core, VPS35 component of Retromer manifest a range of morphological changes in the endo-lysosomal system that resemble those seen in AD.

    I am particularly intrigued by the proteomic finding of enrichment of proteins involved in APP processing, including an accumulation of APP in VPS35 KO lysosomes, as well as an abnormal accumulation of cleaved APP, which suggest that retromer abnormalities may be upstream of Aβ accumulation.

    However, the study has its limitations, especially, the fact that the main model is a neoplastic cell line, which may not fully recapitulate events in normal neurons (although the authors tried to establish the physiological relevance of their model using primary neurons from mouse cortex transfected with plasmids of miRNA Vps35-BFP and observed similar morphological changes).

    I also feel that an opportunity was missed in not isolating extracellular vesicles from the culture supernatant and analyzing their proteome. Since many exosomal proteins were identified by proteomic analysis of this supernatant, it would have been informative to know which proteins are EV-associated and which are not.

    All in all, this is an interesting and potentially impactful study that provides new insights and motivates novel hypotheses.

    View all comments by Dimitrios Kapogiannis

  3. This is a highly comprehensive, cell-based study using multi-omic approaches to understand how Vps35 deficiency leads to the accumulation of lysosomal components. The use of HA-tagged Tmem192 for lysosomal tagging to perform LysoIP is a novel approach that, along with conventional whole-cell proteomics, transcriptomics, and proteomics of biotinylated proteins on the cell surface and in the growth media “secretome,” provides us with a massive dataset for integrated omics-based analysis.

    Among many fascinating findings, it was very interesting to learn that Rab27b is one of the most significantly enriched proteins in the cell and in lysosomes. Rab27b is a critical regulator for the secretion of extracellular vesicles (EV) (Ostrowski et al., 2010), and may also regulate non-exosomal secretion of a-synuclein (Underwood et al., 2020). This study focused on lysosomal accumulation caused by Vps35 deletion, but the dataset also shows enrichment of classical EV markers CD9 and CD81 in lysosomes, CD9 in growth media, and multiple other tetraspanins (TSPAN4, TSPAN6) on the cell surface. These data suggest enhanced secretion of not only lysosomes but also conventional EV and autophagosomal intermediates.

    The protein composition of the lysosome of Vps35 KO cells also show significant enrichment of APP C-terminal fragments and APP processing enzymes, which is also found in brain-derived EVs (Perez-Gonzalez et al., 2012; Muraoka et al., 2020). TSPAN6 is increased in Alzheimer’s disease brain and plays a role in sorting of the C-terminal fragment to the exosome-mediated secretion (Guix et al., 2017), and may also be involved in the accumulation of the C-terminal fragment in the lysosome of Vps35 KO cells. Considering the striking overlap of the pathogenic proteins found in the dysfunctional lysosome and dystrophic neurites, the study suggests a critical role for Vps35 in dystrophic neurite formation surrounding amyloid plaques, as seen in human AD brain and mouse models of amyloidosis.

    References:

    Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, Moita CF, Schauer K, Hume AN, Freitas RP, Goud B, Benaroch P, Hacohen N, Fukuda M, Desnos C, Seabra MC, Darchen F, Amigorena S, Moita LF, Thery C. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol. 2010 Jan;12(1):19-30; sup pp 1-13. Epub 2009 Dec 6 PubMed.

    Underwood R, Wang B, Carico C, Whitaker RH, Placzek WJ, Yacoubian TA. The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein. J Biol Chem. 2020 Jun 5;295(23):8005-8016. Epub 2020 Apr 29 PubMed.

    Perez-Gonzalez R, Gauthier SA, Kumar A, Levy E. The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space. J Biol Chem. 2012 Dec 14;287(51):43108-15. PubMed.

    Muraoka S, DeLeo AM, Sethi MK, Yukawa-Takamatsu K, Yang Z, Ko J, Hogan JD, Ruan Z, You Y, Wang Y, Medalla M, Ikezu S, Chen M, Xia W, Gorantla S, Gendelman HE, Issadore D, Zaia J, Ikezu T. Proteomic and biological profiling of extracellular vesicles from Alzheimer's disease human brain tissues. Alzheimers Dement. 2020 Jun;16(6):896-907. Epub 2020 Apr 17 PubMed.

    Guix FX, Sannerud R, Berditchevski F, Arranz AM, Horré K, Snellinx A, Thathiah A, Saido T, Saito T, Rajesh S, Overduin M, Kumar-Singh S, Radaelli E, Corthout N, Colombelli J, Tosi S, Munck S, Salas IH, Annaert W, De Strooper B. Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments. Mol Neurodegener. 2017 Mar 10;12(1):25. PubMed.

    View all comments by Tsuneya Ikezu

  4. This is a very informative study that catalogues, in great detail, how deficiency of the retromer impacts function of the endolysosomal system and how this relates to maintaining cellular homeostasis.

    Although the work is largely undertaken in cancer cell types, such as HeLa and H4 epithelial cells that were originally isolated from the brain of a patient with neuroglioma, the work provides important new knowledge and strong evidence how disruption of the retromer could cause loss of function and death of neuron and other brain cells. 

    Most importantly, the authors define a new set of specific biomarkers and a fingerprint signature, including increased levels of Rab27b, that could be used to help diagnose people whose neurodegeneration is driven through this pathway.

    Such markers could also be used to study the cellular efficacy of compounds that boost retromer activity, which I understand are currently being developed. 

    In future work, it would be interesting to extend some of these studies to iPSC-derived brain cells.

    View all comments by Dario Alessi

  5. The retromer complex, a critical regulator of membrane protein recycling through the endolysosomal system, has attracted considerable interest both for its likely involvement in Alzheimer’s and Parkinson’s disease pathogenesis, and as a potential therapeutic pathway for disease modification. The authors report here on a highly informative series of targeted proteomic studies of changes in the endolysosomal system following loss of retromer function, as well as accompanying transcriptional changes—in this case in a glioma cell line. Retromer function was disrupted by knocking out one of its core proteins, VPS35, resulting in enlarged intracellular vesicles that appear to be fused endosomes and lysosomes. To map the protein content of these abnormal endolysosomes, tagged lysosome proteins were expressed in the cell lines to facilitate lysosome purification for quantitative proteomics, comparing the retromer-disrupted cells with their isogenic control parental counterparts.

    As well as reinforcing the importance of the retromer in maintaining the integrity of the endolysosomal system, this study uncovers an important role for retromer in lysosome reformation following autophagy. One net effect of the changes following retromer loss of function was increased lysosomal exocytosis, with extracellular secretion of inefficiently degraded cellular material, including key neurodegeneration-associated proteins such as APP, which may contribute to disease progression by promoting intercellular transfer of pathogenic protein forms.

    As a contribution to our increasing understanding of how endolysosome dysfunction can contribute to neurodegenerative disease pathogenesis, this is a very interesting study in its own right. Equally, this study also points to the potential power of applying these robust quantitative approaches to understanding endolysosome dysfunction within the nervous system—for example, in neurons and microglia expressing mutations causal for AD in genes such as SORL1, PSEN1 and APP itself, that result in abnormal lysosome function.

    View all comments by Rick Livesey

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