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Jan 27, 2024

Multiomic Analysis Shows Lysosomes Need Retromer to Stay Healthy

09 Jun 2023 The retromer complex controls protein sorting, transport, and

09 Jun 2023

The retromer complex controls protein sorting, transport, and disposal via the endolysosome system. It goes awry in neurodegenerative diseases, but how so remains unclear. Metabolism breaks down in cells lacking retromer, according to a multiomics study led by Peter Cullen and James Daly at the U.K.'s University of Bristol. In the May 29 Nature Communications, they reported that cell membrane proteins are missorted, lysosomes get bloated with undigested cargo, autophagy stalls, and amyloid precursor protein accumulates. This backup prompts lysosomes to dump excess content outside the cell via exocytosis, which might enable cell-to-cell spread of pathogenic proteins, such as Aβ aggregates.

"There is clear evidence linking retromer to a neuroprotective role in neurodegenerative diseases. This manuscript probes the complexity of retromer function and how it aids in neuroprotection," Cullen told Alzforum.

"This exciting new work by the Cullen group has just made large strides in uncovering these mechanisms," commented Jessica Young of the University of Washington, Seattle.

Retromer regulates endosomal recycling to the cell surface and their retrograde movement to the Golgi. Mutations in the core retromer protein VPS35 have been linked to AD and Parkinson's disease (Rovelet-Lecrux et al., 2015; Jul 2011 news). Retromer is depleted in AD brain, postmortem, while in cell and animal models deficiency of the complex correlates with worse Aβ and tau pathology (Small et al., 2005; Sullivan et al., 2011; Carosi et al., 2020).

To systematically study what goes wrong when retromer is disabled, co-first authors Daly and Chris Danson knocked out VPS35 in human neuroglioma cells, then stained for lysosomes with LAMP1 and for early endosomes with EEA1. Both organelles had swollen and become distorted in the knockout cells. Transmission electron microscopy showed enlarged endolysosomes stuffed with undigested bits of membrane (see image below). The latter was reminiscent of bloated endolysosomes in AD, PD, and Lewy body dementia (Cataldo et al., 2000; Shahmoradian et al., 2019; Crews et al., 2010).

Cullen, and commentator Julia TCW of Boston University, were surprised that targeting a single retromer protein would cause such a striking phenotype. Live cell imaging of the knockouts captured occasional and sluggish splitting of large autophagic lysosomes. This process returns the organelles to their original size, explaining the numerous swollen organelles.

Enlarged Endolysosomes. Normal lysosomes (red arrows) and endosomes (blue arrows) are small and round (left). Retromer knockout cells (middle) contained huge, distorted endolysosomes (yellow arrows) filled with debris (right). [Courtesy of Daly et al., Nature Communications, 2023.]

What was going wrong within these lysosomes? The scientists analyzed proteomes of whole cells and of isolated lysosomes. Within the former, 477 lysosome-associated proteins were enriched, and 246 were depleted. Many hydrolytic enzymes, such as proteases, lipases, and nucleases, were diminished, suggesting a stunted ability to degrade cellular waste.

The VSP35-negative lysosomes were stacked with multiple hydrolytic Rab GTPases, which regulate lysosomal exocytosis and their fusion with endosomes and autosomes. The lysosomal proteome also lacked all components of the BORC complex, proteins that move the organelles through the cytosol by latching onto microtubules.

Dimitrios Kapogiannis was particularly intrigued by the accumulation of proteins involved in APP processing and metabolism (comment below). This mirrors a finding seen in mice lacking VPS35 in hippocampal neurons (Jan 2022 news).

Other enriched lysosomal proteins mapped to networks and signaling pathways implicated in AD, PD, and other neurodegenerative diseases (image below). To the authors, these results suggest stagnant, APP-filled lysosomes that have merged with endo- and autosomes yet failed to degrade their contents or return to their original state.

Diseases Diseases. Many proteins enriched in lysosomes fell into pathways associated with neurodegenerative diseases or related processes. [Courtesy of Daly et al., Nature Communications, 2023.]

Stalled lysosomes even affected the cell surface proteome. In VSP35 knockouts, transmembrane proteins were depleted where they belong—at the membrane—yet enriched in lysosomes, suggesting membrane protein recycling had become backed up. On the other hand, lysosomal proteins, such as LAMP1 and the Nieman Pick Type C-linked cholesterol transporter NPCI, did cluster at the cell surface, as did APP, β-secretase 2, and the APP C-terminal fragments BACE produces. The authors believe these shifts in where proteins end up in the cell indicate lysosome exocytosis.

Indeed, measuring the proteome of the cell media revealed high levels of lysosomal proteins, APP, and other substrates of secretases. The authors suspect that lysosome exocytosis ramps up to compensate for waste buildup. This, in turn, might fuel cell-to-cell spread of pathogenic protein aggregates. The scientists think proteins from this "secretome" could be fluid biomarkers for lysosome dysfunction (see Dec 2020 news). "These data suggest enhanced secretion of not only lysosomes but also conventional extracellular vesicles and autophagosomal intermediates," noted Tsuneya Ikezu, Mayo Clinic, Jacksonville, Florida (comment below).

Scott Small of Columbia University, New York, believes that the secretome can inform about crosstalk between cells. "I think the secretome is the most relevant to neurodegenerative diseases because expelled endo-lysosomal contents from neurons would initiate communication with microglia and astrocytes, and we know how this can sometimes backfire," Small said.

RNA Versus Protein. Only 14 proteins (blue text) were down- or upregulated at the transcriptomic and proteomic levels in whole cells. [Courtesy of Daly et al., Nature Communications, 2023.]

Transcriptomics revealed a disconnect between gene expression and protein concentration. Of the hundreds of proteins perturbed in the VPS35 knockouts, only 14 were up- or downregulated at the transcriptional level (image above). This surprised Cullen. Proteomics and transcriptomics do not always correlate tightly. Cullen thinks the disconnect here indicates that the problems caused by retromer knockout lie in protein degradation, not transcription. That genes encoding lysosomal proteins, such as the membrane receptor sortilin, NPC1, and the protease cathepsin D, were upregulated despite not showing up in lysosomes suggests problems with protein trafficking.

All morphological, proteomic, and transcriptomic changes in the retromer knockout endolysosomes were reversed once VPS35 was re-expressed. "This tells us that the changes we're seeing are a true reflection of an on-target effect of retromer perturbation," Cullen said.

To TCW's mind, this makes retromer interesting as a drug target. "Many small molecules activate retromer function, and it will be important to figure out the key up- and downstream proteins or transcription factors that boost retromer function," she told Alzforum (see Jan 2020 news).—Chelsea Weidman Burke

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.

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.

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.

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.

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.

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