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Confocal imaging of a macrophage interacting with aggregated LDL

(J. Cell Sci., 2016. PMID: 26801085, doi: 10.1242/jcs.181743).

This video shows confocal microscopy of the site where a macrophage encounters a large aggregate of LDL that mimics the lipoprotein deposits in atherosclerotic lesions. The lipoprotein is shown in red, the plasma membrane is grey, and F-actin (phalloidin labeling) is shown in green. The macrophage creates deep invaginations containing LDL that are surrounded by actin structures that are essential for formation of the membrane invaginations. We have shown that macrophages secrete lysosomal enzymes to digest the lipoprotein deposits outside the cell.

Role of macrophages in atherosclerosis.

We found that very large atherogenic lipoproteins (β-VLDSs) entered macrophages by alternate endocytic pathways as compared to the classical coated pit pathway followed by LDL. We also found that there was extracellular catabolism of the large β-VLDSs while they were still on the cell surface. We then turned out attention to aggregates of LDL, which are a more accurate model of the atherogenic lipoproteins that macrophages encounter in the wall of blood vessels. We again found that there was catabolism of these lipoproteins outside the cell, and surprisingly the hydrolysis of cholesteryl esters was carried out by lysosomal acid lipase, a lysosomal enzyme that requires a low pH for activity. More recently, we showed that macrophages create an extracellular sealed compartment when they interact with aggregated LDL, which we have called a lysosomal synapse. The cells acidify this compartment by cell surface V-ATPases, and they secrete lysosomal contents into the synapses. This leads to extracellular catabolism of the aggregated LDL, which is eventually internalized after being broken into small pieces. We are exploring other biological contexts in which similar processes are used by macrophages to degrade large objects. The process in macrophages has general similarity to a similar well-characterized function of osteoclasts.

  1. Singh RK, Barbosa-Lorenzi VC, Lund FW, Grosheva I, Maxfield FR, Haka AS. Degradation of aggregated LDL occurs in complex extracellular sub-compartments of the lysosomal synapse. J Cell Sci. 2016 Mar 1;129(5):1072-82. PubMed PMID: 26801085; PubMed Central PMCID: PMC4813320.

  2. Haka AS, Grosheva I, Singh RK, Maxfield FR. Plasmin promotes foam cell formation by increasing macrophage catabolism of aggregated low-density lipoprotein. Arterioscler Thromb Vasc Biol. 2013 Aug;33(8):1768-78. PubMed PMID: 23702659; PubMed Central PMCID: PMC3716867.

  3. Haka AS, Grosheva I, Chiang E, Buxbaum AR, Baird BA, Pierini LM, Maxfield FR. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins. Mol Biol Cell. 2009 Dec;20(23):4932-40. PubMed PMID: 19812252; PubMed Central PMCID: PMC2785736.

  4. Buton X, Mamdouh Z, Ghosh R, Du H, Kuriakose G, Beatini N, Grabowski GA, Maxfield FR, Tabas I. Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL). Prolonged cell-surface contact during which ldl-cholesteryl ester hydrolysis exceeds ldl protein degradation. J Biol Chem. 1999 Nov 5;274(45):32112-21. PubMed PMID: 10542246.

  5. Singh, R.K., Haka, A.S., Brumfield, A., Grosheva, I., Bhardwaj, P., Chin, H.F., Xiong, Y., Hla, T., and Maxfield, F.R. (2017) Ceramide Activation of RhoA/Rho Kinase Impairs Actin Polymerization during Aggregated LDL Catabolism. J. Lipid Res. 58: 1977-1987.

  6. Wang, Y., Subramanian, M., Yurdagul, A. Barbosa-Lorenzi, V.C., Cai, B., de Juan-Sanz, J., Ryan, T.A., Nomura, M., Maxfield, F.R., Tabas, I. (2017) Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages. Cell 171:331-345. doi: 10.1016/j.cell.2017.08.041

  7. 152.Nguyen, A.D.,  Nguyen, T.A.,  Singh, R.K., Eberlé, D., Zhang, J.,  Abate, J.P., Robles, A., Koliwad, S.,  Huang, E.J., Maxfield, F.R., Walther, T.C., Farese, Jr., R.V. (2018) Progranulin in the hematopoietic compartment protects mice from atherosclerosis
    Atherosclerosis 277: 145-154. doi: 10.1016/j.atherosclerosis.2018.08.042; PMID: 30212683; PMCID: PMC6432779.

  8. Singh, R.K., Haka, A.S., Bhardwaj, P., Zha, X., and Maxfield, F.R. (2019) Dynamic actin reorganization and Vav/Cdc42-dependent actin polymerization promote macrophage aggregated LDL (Low-Density Lipoprotein) uptake and catabolism. Arteriosclerosis, Thrombosis, and Vascular Biology 39: 137-149.  doi: 10.1161/ATVBAHA.118.312087, PMID:30580573.  PMCID: PMC6344252.

  9. Singh, R.K., Haka, A.S., Asmal, A, Barbosa-Lorenzi, V.C., Grosheva, I. Chin, H.F., Xiong, Y,. Hla, T, and Maxfield, F.R., (2019) TLR4-dependent signaling drives extracellular catabolism of low-density lipoprotein aggregates. Arteriosclerosis, Thrombosis, and Vascular Biology 40: 86-102. PMID: 31719160, PMCID: PMC6928397
    bioRxiv preprint doi: https://doi.org/10.1101/610162

  10. Maxfield, F.R., Barbosa-Lorenzi, V.C., and Singh, R.K. (2019) Digestive Exophagy: Phagocyte digestion of objects too large for phagocytosis. Traffic 21: 6-12. PMID:31664749; doi: 10.1111/tra.12712

  11. ingh, R.K., Lund, F.W., Haka, A.S., and Maxfield, F.R. (2019) High density lipoprotein or cyclodextrin extraction of cholesterol from aggregated LDL reduces foam cell formation. J. Cell. Sci. doi: 10.1242/jcs.237271, PMCID: PMC6918773

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Intracellular cholesterol transport and development of therapies for NPC disease.

The intracellular transport of cholesterol is poorly understood. We developed microscopy methods to study well-behaved fluorescent sterols and have begun to characterize several steps of cholesterol transport in cells. We demonstrated that there is rapid non-vesicular transport of sterol between the plasma membrane and the ERC, and we have begun to characterize the role of a specific sterol binding protein, StarD4, in this transport. A biologically important step in sterol transport is the efflux of lipoprotein-derived cholesterol from late endosomes and lysosomes – process that does not occur in cells from patients with Niemann Pick disease type C (NPC). Using our expertise in optical imaging, we developed a high throughput image-based screen to identify small molecules that could correct the cholesterol storage in NPC cells. We found that histone deacetylase (HDAC) inhibitors, including an FDA-approved drug, Vorinostat, could correct the defect in many NPC1 mutant cells. We are now participating in a clinical trial funded by a U01 grant.

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  1. Iaea DB, Dikiy I, Kiburu I, Eliezer D, Maxfield FR. STARD4 Membrane Interactions and Sterol Binding. Biochemistry. 2015 Aug 4;54(30):4623-36. PubMed PMID: 26168008; PubMed Central PMCID: PMC4527246.

  2. Praggastis M, Tortelli B, Zhang J, Fujiwara H, Sidhu R, Chacko A, Chen Z, Chung C, Lieberman AP, Sikora J, Davidson C, Walkley SU, Pipalia NH, Maxfield FR, Schaffer JE, Ory DS. A murine Niemann-Pick C1 I1061T knock-in model recapitulates the pathological features of the most prevalent human disease allele. J Neurosci. 2015 May 27;35(21):8091-106. PubMed PMID: 26019327; PubMed Central PMCID: PMC4444535.

  3. Mesmin B, Pipalia NH, Lund FW, Ramlall TF, Sokolov A, Eliezer D, Maxfield FR. STARD4 abundance regulates sterol transport and sensing. Mol Biol Cell. 2011 Nov;22(21):4004-15. PubMed PMID: 21900492; PubMed Central PMCID: PMC3204063.

  4. Pipalia NH, Cosner CC, Huang A, Chatterjee A, Bourbon P, Farley N, Helquist P, Wiest O, Maxfield FR. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5620-5. PubMed PMID: 21436030; PubMed Central PMCID: PMC3078401.

  5. Pipalia, N.H., Subramanian, K., Mao, S., Ralph, H., Hutt, D.M., Scott, S.M., Balch, W.E., and Maxfield, F.R. (2017 ) Histone deacetylase inhibitors correct the cholesterol storage defect in most NPC1 mutant cells. J Lipid Res 58: 695-708. PMID: 28193631

  6. Iaea, D.B., Mao, S., Lund, F.W., and Maxfield, F.R. (2017) Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane. Mol Biol Cell. 28: 1111-1122.  PMID: 28209730

RESEARCH

We are interested both in the basic mechanisms regulating the movement of molecules through cells as well as the role that these processes play in specific diseases. We use fluorescent tracers to follow the fates of specific molecules. For example, we have used naturally fluorescent sterols to see how cholesterol moves around in cells. We also use various biochemical and molecular biology techniques to test the role of specific proteins in directing membrane traffic in living cells. We are also studying disease-related endocytic processes such as the uptake of large lipoproteins by macrophages leading to atherosclerotic lesions and the interaction of cells with the proteins that form Alzheimer's disease plaques. Each of these uses variations of the basic endocytic processes that we have characterized.

Endocytic pH regulation and amyloid degradation.

A longstanding interest is the acidification of endocytic organelles and the biological consequences of this acidification. A particular example is degradation of Alzheimer’s Aβ by microglia. We had shown that fibrillar Aβ is internalized into microglia by receptor mediated endocytosis, but it is degraded ineffectively. We showed that this was due to poor acidification of the microglial lysosomes. Activation of microglia with MCSF led to increased acidification of lysosomes and rapid degradation of internalized fibrillar Aβ. We also showed that the enhanced acidification required delivery of a chloride transporter, Clc-7, to late endosomes and lysosomes.

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a.     Paresce, D.M., Chung, H., and Maxfield, F.R. (1997) Slow degradation of aggregates of the Alzheimer’s disease amyloid b-protein by microglial cells.  J. Biol. Chem. 272: 29390-29397. PMID: 9361021

b.     Chung, H., Brazil. M.I., Soe, T.T., and Maxfield, F.R. (1999) Uptake, degradation and release of fibrillar and soluble forms of Alzheimer’s amyloid -peptide by microglial cells, J. Biol. Chem. 274: 32301-32308.  PMID: 10542270

c.     Majumdar A, Cruz D, Asamoah N, Buxbaum A, Sohar I, Lobel P, Maxfield FR. Activation of microglia acidifies lysosomes and leads to degradation of Alzheimer amyloid fibrils. Mol Biol Cell. 2007 Apr;18(4):1490-6. PubMed PMID: 17314396; PubMed Central PMCID: PMC1838985.

d.     Majumdar, A., Chung, H., Dolios, G., Wang, R., Asamoah, N., Lobel, P., and Maxfield, F.R. (2008) Degradation of fibrillar forms of Alzheimer’s amyloid eta-peptide by macrophages, Neurobiology of Aging 29: 707-715. PMCID: PMC2424018

e.     Majumdar A, Capetillo-Zarate E, Cruz D, Gouras GK, Maxfield FR. Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes. Mol Biol Cell. 2011 May 15;22(10):1664-76. PubMed PMID: 21441306; PubMed Central PMCID: PMC3093319.

f.      Solé-Domènech, S., Cruz, D.L., Capetillo-Zarate, E., and Maxfield, F.R. (2016) The endocytic pathway in microglia during health, aging and Alzheimer's disease. Ageing Res Rev, S1568-1637(16)30142-8.   PMID: 27421577; doi: 10.1016/j.arr.2016.07.002. 

G. Solé-Domènech., Rojas, A.V., Maisuradze, G.G., Scheraga, H.A., Lobel, P., and Maxfield, F.R. (2018) The lysosomal enzyme tripeptidyl peptidase 1 destabilizes fibrillar amyloid-beta by multiple endoproteolytic cleavages within the -sheet domain. Proc. Nat. Acad. Sci., USA, : 1493-1498. PMID: 29378960PMCID: PMC5816203.

Macrophage clearance of apoptotic adipocyte

We have made notable progress on this field by showing that macrophages form acidic, hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments contain lysosomal enzymes, which are delivered by localized exocytosis. The degradation of dead adipocyte fragments culminates in macrophage foam cell formation. We have also recently described the importance of this process in vivo, and we showed that a defect in macrophage lysosome exocytosis leads to dead adipocyte accumulation, culminating in diet-induced obesity features such as insulin resistance, hepatosteatosis and visceral lipoatrophy. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages.

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https://www.ncbi.nlm.nih.gov/pubmed/27044658

https://www.ncbi.nlm.nih.gov/pubmed/28954231

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