Exosomes Divulge Lysosomal Unrest, Signal AD?

Neurons expel vesicles called exosomes when multivesicular bodies fuse with the plasma membrane and release these tiny packets. Exosomes rid cells of waste, carry messages between them, and can cross the blood-brain barrier. Scientists led by Edward Goetzl, University of California, San Francisco, and Dimitrios Kapogiannis, National Institute on Aging, Baltimore, have developed a method to capture neuron-derived exosomes in blood and are exploring their potential as disease markers. Previously they reported that exosome levels of pathogenic proteins, insulin-resistance markers, and cell-survival proteins distinguished people with Alzheimer’s from controls (Aug 2014 news; Fiandaca et al., 2014; Kapogiannis et al., 2014). In the July 7 Neurology, the group now reports differences in lysosomal proteins, as well. A small set distinguished patients from controls up to 10 years before clinical diagnosis, introducing them as new candidates for early disease detection, wrote the authors.

“It’s refreshing to see the biomarker search expand to emphasize another pathophysiology that is prominent in AD,” said Ralph Nixon, New York University School of Medicine, who was not involved in the study. “This could be the tip of the iceberg in terms of other exosomal markers that reflect endosomal, lysosomal, and autophagy pathology.”

Lysosomes are acidic vesicles inside cells that digest unwanted material. The lysosomal network in neurons clogs up and malfunctions in AD and other disorders (for a review, see Ihara et al., 2012). When lysosomes stop breaking down garbage, the cell has to get rid of it in other ways, one of which may be to eject it in exosomes (Alvarez-Erviti et al., 2011). Could the contents of neurally derived exosomes offer a window on lysosomal dysfunction?

To answer this question, Goetzl and colleagues conducted a two-part study. In a cross-sectional portion, they took blood from 26 patients with mild cognitive impairment (MCI), eight with AD dementia, and 16 with frontotemporal dementia to compare the lysosomal proteins in their neuron-derived exosomes to each other and those of age-matched controls. Patients with AD were diagnosed by either Dubois or NINCDS-ADRDA criteria, with Clinical Dementia Rating (CDR) global scores of at least 1.0 (Dubois et al., 2007; McKhann et al., 1984). MCI patients were diagnosed according to Petersen criteria and had CDR global scores of 0.5 (Petersen, 2004). The scientists examined another 20 AD patients longitudinally. These people gave blood twice: once when they were cognitively intact—one to 10 years before disease onset—and again once they were symptomatic.

From those blood samples, the researchers isolated neuronal exosomes using an antibody for L1CAM, a neural adhesion protein highly expressed in the central nervous system. They then measured the levels of lysosomal proteins inside, including total protein with a ubiquitin tag that targets waste to lysosomes; lysosome-associated membrane protein 1 (LAMP-1), the lysosomal aspartyl endoproteinase cathepsin D, and the chaperone heat-shock protein 70 (HSP70).

In this cross-sectional data, there was some overlap in protein levels between groups. Even so, average cathepsin D and total ubiquitinylated protein levels were twice as high in the AD group as in controls, whereas HSP70 was lower. AD patients had more LAMP-1 on average, but values between groups overlapped so much that protein levels did not distinguish AD patients. FTD followed the same trend, though to a lesser extent. The combination of cathepsin D, total ubiquitin, and HSP70 distinguished all AD patients from controls, and 96 percent of AD from FTD patients. Cathepsin D and HSP70 likewise distinguished all FTD patients from controls. Researchers who were not involved in this study interpreted this result cautiously. They noted that the error rate inherent in clinical diagnosis makes perfect distinction by a biomarker unlikely, and called for replication of the data.

In the longitudinal data, all four lysosomal proteins separated AD patients from controls. What’s more, levels started changing when the study participants were still cognitively normal, hinting that the underlying process happens years before disease onset.

The results suggest that neuronal lysosome proteins in exosomes could become useful biomarkers for preclinical AD, the authors believe. Goetzl suspects the proteins may be abnormal even earlier than 10 years before diagnosis. He and Kapogiannis have started mining data from 400 people in the Baltimore Longitudinal Study on Aging, who gave blood regularly 40 to 50 years before getting Alzheimer’s disease. He predicts a shift in lysosomal proteins in middle age.

The authors acknowledged the small sample size in this study. Goetzl said ongoing studies include observation of exosomal profiles in healthy aging, their ability to predict conversion from MCI to dementia, and correlation with imaging markers and cerebrospinal fluid proteins.

Kaj Blennow, University of Gothenburg, Sweden, noted that certain peripheral cells produce L1CAM, so the assay may extract peripheral exosomes as well (see full comment below). Goetzl conceded that the vesicles are likely not 100 percent neuronal; he estimated based on enrichment of other neuronal markers that 80 percent are.

Can these exosomal markers distinguish diseases that are each tied to lysosomal dysfunction? Nixon said this might be achieved by looking for additional lysosome markers in exosomes, or for disease-specific markers. Exosomal α-synuclein, Aβ, or p-tau could yield disease-specific profiles for Parkinson’s or Alzheimer’s, he said. —Gwyneth Dickey Zakaib

Comments

    • User Profile Image Kaj Blennow
      University of Goteborg, Sahlgrenska University Hospital
    • Posted:

    This is an interesting study with large potential, since it identifies a set of candidate AD biomarkers in blood exosomes. The principle of isolating exosomes and quantifying specific candidate protein biomarkers is an attractive approach to identifying novel AD biomarkers. Indeed, the identified biomarker candidates cathepsin D, LAMP-1, ubiquitinylated proteins, and HSP70 fit well with the recent interest in the relevance of a autophagocytic-lysosomal dysfunction in AD.

    The results need to be validated in larger cohorts. However, a concern is how certain we can be that these "neural-derived plasma exosomes” are derived from the brain. To pull out (or affinity-purify) neural-derived exosomes from plasma, an antibody against CD171 (L1CAM neural adhesion protein) called clone 5G3 was used. However, CD171 is also expressed on blood cells (monocytes and lymphocytes), blood vessels, the peripheral nervous system, collecting tubules of the kidney and in many types of tumor (Huszar et al., 2006; Felding-Habermann et al., 1997). Even if this peripheral expression may not limit the use of plasma exosome proteins as AD biomarkers, it may act as a confounder when aiming at linking plasma exosome levels (or protein levels in plasma exosomes) to brain molecular pathology, and introduce noise due to, for example, peripheral inflammation or other conditions. Nevertheless, the findings are clearly interesting and warrant replication in independent studies.

    References:

    Huszar M, Moldenhauer G, Gschwend V, Ben-Arie A, Altevogt P, Fogel M. Expression profile analysis in multiple human tumors identifies L1 (CD171) as a molecular marker for differential diagnosis and targeted therapy. Hum Pathol. 2006 Aug;37(8):1000-8. Epub 2006 Jun 21 PubMed.

    Felding-Habermann B, Silletti S, Mei F, Siu CH, Yip PM, Brooks PC, Cheresh DA, O'Toole TE, Ginsberg MH, Montgomery AM. A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins. J Cell Biol. 1997 Dec 15;139(6):1567-81. PubMed.

    View all comments by Kaj Blennow

  2. Goetzl et al. show that Alzheimer’s disease and frontotemporal dementia patients have differences in biomarkers located within blood exosomes secreted by brain neuronal cells. These biomarker differences could potentially become diagnostic for these diseases, and thus this is an important paper. While the use of exosomes as a potential diagnostic pool is not new (e.g., Kang et al., 2014), this is among the first publications to use neuronally derived exosomes to diagnose dementias, and as such is of particular interest to the readers of this forum.

    Exosomes come from the cytosol of cells and thus exosome analysis is of the cellular cytosolic composition. Current methods of probing brain composition in patients rely on cerebrospinal fluid, which only allows study of the composition of the extracellular space. As such, this paper opens new opportunities for evaluating the intracellular composition of neurons and glial cells in patients. This has many potential applications, such as estimating the dose of a therapeutic agent in neurons rather than only in the extracellular space, as is the case now.

    Some key findings of the paper are that cathepsin D (CatD) and lysosome-associated membrane protein 1 (LAMP1) levels are significantly elevated and heat-shock protein 70 (HSP70) is reduced in neuronally derived exosomes from AD patients relative to controls. These data are important because they show that in AD patients, there is lysosomal breakdown and cathepsin D release to the cytoplasm. That is direct support for the “calpain-cathepsin” cell-death hypothesis of AD (Yamashima, 2013), which is that calpain activation reduces HSP70, causing lysosomal membrane permeabilization and the release of lysosomal CatD (as well as cathepsin B and L) to the cytoplasm, where its proteolytic activity results in cell death and inflammation of AD. A practical application of these findings is that there are now markers that can be used to evaluate in patients the potential of therapeutic compounds to affect this form of cell death.

    References:

    Kang GY, Bang JY, Choi AJ, Yoon J, Lee WC, Choi S, Yoon S, Kim HC, Baek JH, Park HS, Lim HJ, Chung H. Exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related macular degeneration. J Proteome Res. 2014 Feb 7;13(2):581-95. Epub 2014 Jan 16 PubMed.

    Yamashima T. Reconsider Alzheimer's disease by the 'calpain-cathepsin hypothesis'-A perspective review. Prog Neurobiol. 2013 Jun;105:1-23. PubMed.

    View all comments by Greg Hook

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References

News Citations

  1. Exosomes Stand Out as Potential Blood Biomarkers

Paper Citations

  1. Fiandaca MS, Kapogiannis D, Mapstone M, Boxer A, Eitan E, Schwartz JB, Abner EL, Petersen RC, Federoff HJ, Miller BL, Goetzl EJ. Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study. Alzheimers Dement. 2014 Aug 14; PubMed.
  2. Kapogiannis D, Boxer A, Schwartz JB, Abner EL, Biragyn A, Masharani U, Frassetto L, Petersen RC, Miller BL, Goetzl EJ. Dysfunctionally phosphorylated type 1 insulin receptor substrate in neural-derived blood exosomes of preclinical Alzheimer's disease. FASEB J. 2015 Feb;29(2):589-96. Epub 2014 Oct 23 PubMed.
  3. Ihara Y, Morishima-Kawashima M, Nixon R. The ubiquitin-proteasome system and the autophagic-lysosomal system in Alzheimer disease. Cold Spring Harb Perspect Med. 2012;2(8) PubMed.
  4. Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, Wood MJ, Cooper JM. Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol Dis. 2011 Jun;42(3):360-7. PubMed.
  5. Dubois B, Feldman HH, Jacova C, Dekosky ST, Barberger-Gateau P, Cummings J, Delacourte A, Galasko D, Gauthier S, Jicha G, Meguro K, O'brien J, Pasquier F, Robert P, Rossor M, Salloway S, Stern Y, Visser PJ, Scheltens P. Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria. Lancet Neurol. 2007 Aug;6(8):734-46. PubMed.
  6. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984 Jul;34(7):939-44. PubMed.
  7. Petersen RC. Mild cognitive impairment as a diagnostic entity. J Intern Med. 2004 Sep;256(3):183-94. PubMed.

Further Reading

Papers

  1. De Toro J, Herschlik L, Waldner C, Mongini C. Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front Immunol. 2015;6:203. Epub 2015 May 4 PubMed.
  2. Coleman BM, Hill AF. Extracellular vesicles--Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases. Semin Cell Dev Biol. 2015 Apr;40:89-96. Epub 2015 Feb 20 PubMed.
  3. Rajendran L, Bali J, Barr MM, Court FA, Krämer-Albers EM, Picou F, Raposo G, van der Vos KE, van Niel G, Wang J, Breakefield XO. Emerging roles of extracellular vesicles in the nervous system. J Neurosci. 2014 Nov 12;34(46):15482-9. PubMed.

Primary Papers

  1. Goetzl EJ, Boxer A, Schwartz JB, Abner EL, Petersen RC, Miller BL, Kapogiannis D. Altered lysosomal proteins in neural-derived plasma exosomes in preclinical Alzheimer disease. Neurology. 2015 Jul 7;85(1):40-7. Epub 2015 Jun 10 PubMed.

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