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.

  • In cells lacking retromer protein VSP35, endolysosomes bloat up with surface membrane proteins.
  • Autophagy stalls, APP accumulates, and lysosome exocytosis ramps up.
  • Lysosomal, cell surface, and secreted proteomes become perturbed.

“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


  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.


    . Pharmacological chaperones stabilize retromer to limit APP processing. Nat Chem Biol. 2014 Jun;10(6):443-9. Epub 2014 Apr 20 PubMed.

    . 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.

    . 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.

  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.

  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.


    . 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.

    . 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.

    . 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.

    . 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.

    . 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.

  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.

  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.

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News Citations

  1. Sorting Out Parkinson’s: Exome Sequencing Points to Recycling Defect
  2. Neuron-Specific Retromer Identified—Does It Stave Off Alzheimer’s?
  3. Biomarkers of Errant Endosome Spotted in Cerebrospinal Fluid
  4. Chaperone Stabilizer Fends Off Amyloid, Memory Loss in Mice

Paper Citations

  1. . De novo deleterious genetic variations target a biological network centered on Aβ peptide in early-onset Alzheimer disease. Mol Psychiatry. 2015 Sep;20(9):1046-56. Epub 2015 Jul 21 PubMed.
  2. . Model-guided microarray implicates the retromer complex in Alzheimer's disease. Ann Neurol. 2005 Dec;58(6):909-19. PubMed.
  3. . Retromer disruption promotes amyloidogenic APP processing. Neurobiol Dis. 2011 Aug;43(2):338-45. PubMed.
  4. . Retromer regulates the lysosomal clearance of MAPT/tau. Autophagy. 2020 Sep 22;:1-21. PubMed.
  5. . Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
  6. . Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes. Nat Neurosci. 2019 Jul;22(7):1099-1109. Epub 2019 Jun 24 PubMed.
  7. . Selective molecular alterations in the autophagy pathway in patients with Lewy body disease and in models of alpha-synucleinopathy. PLoS One. 2010;5(2):e9313. PubMed.

Further Reading

Primary Papers

  1. . Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health. Nat Commun. 2023 May 29;14(1):3086. PubMed.