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Lysosomal stress: a new player in perturbed lipid metabolism

Gabriel, T.L.

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Citation for published version (APA):Gabriel, T. L. (2017). Lysosomal stress: a new player in perturbed lipid metabolism.

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Download date: 29 Jun 2020

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GENERAL DISCUSSION

Recently, lysosomes re-entered the spotlight as key subcellular organelles which carry out an important role in nutrient sensing and signaling pathways involved in cell growth and metabolism. During starvation lysosome number and autophagic activity increase, resulting in protein, carbohydrate and lipid degradation providing energy to the organism. A new important development in the field was the discovery of a gene network named CLEAR (Coordinated Lysosomal Expression and Regulation) by Ballabio and co-workers. The CLEAR network regulates lysosomal biogenesis and activity, as well as other lysosomal related functions including exo- and endocytosis, autophagy, phagocytosis and immune response 1–3. MITF, TFEB and TFE3 are the master regulators of CLEAR network, those transcription factors belong to the MiT family characterized by their Basic helix-loop-helix leucine zipper domain 4. When nutrients are abundant, mTORC1 is active and MITF, TFEB and TFE3 are sequestered predominantly outside the nucleus, resulting in the inhibition of their transcription function. Consequently lysosomal formation and autophagy induction are prevented. On the contrary, the inhibition of mTORC1 by starvation or inhibitors, stimulates MiT members nuclear localization and activation followed by boosted autophagy and lysosome biogenesis. In 2013 Settembre et al. proposed a model of TFEB regulation and lysosomal biogenesis in which mTORC1 which was previously described to be located on the surface of the lysosome by Sabatini group is together with other protein complexes able to sense the nutrient status named lysosomal nutrient sensing machinery (LYNUS) 2,5. When nutrients are at low levels, LYNUS sense it and mTORC1 is inhibited, promoting MiT activation and translocation from the cytosol to the nucleus to start the CLEAR transcription program 6. Thus, lysosomes have a critical role in cell metabolism. They are able to sense nutrient status and regulate signaling pathways involved in cell metabolism, and they are the catabolic machinery of the cell providing for nutrients under starving conditions. Complex lipids, like sphingolipids are degraded within the lysosomal acidic compartment and consequently lysosomes are involved in regulating their level. Considering that sphingolipids are bioactive signaling molecules involved in the modulation of inflammation, cell growth, apoptosis and survival, and that lysosomal activity can regulate sphingolipid levels, lysosomal activity can have an impact on different cellular processes governed by sphingolipids.

Unbalanced lipid homeostasis induces lysosomal stress: Gpnmb as a lysosomal stress marker

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Any distress disrupting normal endosome-lysosomal activity may affect cellular lipid levels since most of the lipid-related molecules are catabolized within this system. Lysosomal storage disorders (LSD) are an excellent example of how a mutation in a lysosomal protein generates accumulation of undigested material within the cell, contributing to further lysosomal dysfunction. As a consequence of this undigested material accumulation other lysosomal metabolic pathways get affected as well and in many instances unanticipated secondary metabolites are generated which may accumulate as well. Considering that lysosomes present a sensitive lysosome-to-nucleus signaling mechanism, it was expected that LSD impact lysosomal biogenesis and autophagy 7. In this thesis it is shown that the autophagy related protein glycoprotein nonmetastatic melanoma protein b (Gpnmb) was strongly induced at the gene and protein level in macrophages in the LSD gaucher disease (GD) and niemann pick-C (NPC) disease (chapter 2 and chapter 3). In an in vitro setting using the macrophage-like cell line RAW264.7, Gpnmb was induced by the NPC mimicking agent U18666A, the lysosomal stressors inhibiting vacuolar H+-ATPase such as chloroquine (CQ), concanamycin A and bafilomycin, as well as by palmitate (chapter 3 and chapter 5) . Although the cause of GD and NPC disease is different, patients share to some extent phenotypic features like the presence of lipid-laden macrophages, increase of SLs, hepatosplenomegaly and neurodegeneration, suggesting that lysosomal perturbation drives the phenotype of those conditions and not only the mutation of specific enzymes. Furthermore, obesity is associated with altered SL metabolism and Gpnmb was also strongly induced by lipid-overload in ATM as it occurs during obesity (Chapter 5) (figure 1).

Interestingly, LDLR knockout mice fed a western-type diet, presented a steatotic liver with a strong induction of Gpnmb. Upon treatment with the GlcCer synthase inhibitor AMP-DNM the effect of the western-type diet on various metabolic read outs was normalized. Importantly, also Gpnmb expression was reduced, suggesting a role for GlcCer, or a derivative in regulating Gpnmb expression 8. This was further supported in RAW264.7 cells when U18666A was used to mimick the NPC phenotype. Also in this case treatment with the glucosylceramide synthase (GCS) inhibitor N-butyl-1 deoxybojirimycin (NB-DNJ, Zavesca®), the only EMA approved medicine for NPC treatment, Gpnmb was reduced. Interestingly, cholesterol levels remained unaltered, suggesting that Gpnmb induction is driven by GLs rather than by cholesterol accumulation (chapter 3). The mechanistics behind lysosomal stress and Gpnmb induction in GD and NPC are incompletely understood. However the results presented in this thesis strongly suggest that a GLs may play a role in regulation Gpnmb expression.

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In line with the observation of Gpnmb induction by lysosomal stress other lysosomal stressors of different nature like; CQ and the buffer molecule like HEPES were investigated (chapter 2, chapter 3 and chapter 4). Classically, HEPES has been considered a Good’s Buffer suitable for biological and biochemical research 9. Good’s buffer compounds were described around the sixties and have to meet several criteria among others high solubility in water, membrane impermeable and biochemical inertness 9. Strikingly, culture of RAW264.7 in presence of HEPES revealed a great impact on lysosomes and immunity, which can be summarized as follows: more vacuolar structures were present, Gpnmb and other lysosomal markers were strongly induced suggesting lysosomal stress, autophagic induction and potentiates M1/M2 response (chapter 4). The complete mechanism of HEPES on lysosomal stress induction is not fully understood, but seems to occur independently of mTORC1, requires MiT, but deserves continued investigation given its widespread use and possible impact on data interpretation, therefore it is advised to use it with caution.

In the context of obesity, were lipid imbalance also occurs, hence more free fatty acids are accumulated than can be degraded, an increase in lysosomal biogenesis is also found. We show that palmitate coupled to BSA, CQ and mTORC1 inhibition through torin-1 also induced Gpnmb in a MITF-dependent fashion (Chapter 5). Yet the nature of these different stimuli and subsequent MITF activation might be different. CQ is a lysomotropic agent able to diffuse and cross cellular membranes, accumulating within the lysosome and increasing its pH. Palmitate coupled to BSA enters the lysosome in a pinocytosis and caveolae-dependent manner 10,11. Like CQ, it has been show that palmitate increases lysosomal pH in INS1 and primary pancreatic cells 12. In line with those observations, it has been shown in numerous studies that palmitate can induce lysosomal membrane permeabilization (LMP), generating pores in the lysosomal membrane causing the release to the cytosol of some cathepsins 13,14. The mechanism underlying the palmitate-LMP formation is not well understood and still remains to be elucidated whether the lysosomal palmitate-dependent pH increase is caused by LMP or through another mechanism. Interestingly, BSA coupled oleate did not induce Gpnmb, suggesting that palmitate effects may relate to its chemical structure (Chapter 5). Other reports also reflected on opposite inflammatory outcome in macrophages. Palmitate induces a more pro-inflammatory polarization in macrophage, whereas oleate promotes an anti-inflammatory phenotype 15,16. Most likely again the nature of their chemical structure contributes to this. Palmitate is a more rigid molecule, whereas oleate containing a double bound is more flexible. In this context Leekumjorn et al.

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showed using a computational approach and liposomes that palmitate causes a greater membrane destabilization resulting in a larger leakage than oleate 17. Furthermore, we demonstrated that only palmitate increases ceramide de novo synthesis in RAW264.7 (Chapter 5). Interestingly, when RAW264.7 cells were loaded with palmitate in the presence of myriocin, a ceramide de novo synthesis inhibitor, Gpnmb induction was not prevented, even slightly induced. This suggests that ceramides are not the drivers of lysosomal stress in palmitate laden cells (Chapter 5).

Figure 1. Similarities and differences between obese ATM and a lipid storage disease macrophage. Both macrophage types have in common a foamy phenotype due to the accumulation of lipid droplets. Also in insulin resistance, sphingolipid levels and lysosomal stress markers are elevated. The obese ATM phenotype is generally driven by environmental factors, due to over-nutrition and sedentary conditions, promoting an increase in adipose tissue and ATM recruitment and M1 polarization. On the contrary, lipid storage macrophage phenotype is induced by a genetic deficiency, the bone marrow present a macrophage infiltration, however there is no clear polarization towards an M1/M2 phenotype 18–23 .

Pleiotropic Gpnmb biological function

Immunohistochemistry analysis of Gpnmb expression in liver and spleen from NPC

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mice, and WAT in ObOb mice showed that Gpnmb induction occurs predominantly in macrophages (Chapter 3 and chapter 5). Gpnmb is induced by different lysosomal stressors namely CQ, U18666A, HEPES, sucrose, Torin-1 and bafilomicyn A as well as by physiological components like palmitate. In this thesis we show that chloroquine, torin-1 and palmitate Gpnmb induction is MITF/TFEB dependent in Raw264.7 (Chapter 4 and chapter 5). Interestingly, TFEB is ubiquitously found in different tissues and cell types, whereas MITF expression is more confined to specific cells like macrophages, B cells, osteoclast, melanocytes, NK cells, mast cells and some neuronal cells 24,25. It has been shown that TFEB and MITF can generate homo- and heterodimers that can bind to specific DNA sequences to start their transcription program 26. Furthermore it has been shown that they play redundant function in osteoclast development 26. Combining the fact that they can form heterodimers with the fact that they have different splice variants, allows a high degree of variations in dimer diversity alongside with different possible targets, providing a possible explanation why Gpnmb expression only occurs in specific cell types and is not ubiquitously expressed 4,24,26,27. Despite that Gpnmb has a restricted expression profile and a not fully understood function, it is a highly conserved across species 28. Regarding its function , Gpnmb has recently been described to be expressed in astrocytes and contributes to attenuation of neurotoxicity in amyotrophic lateral sclerosis (ALS) 29. The specific mechanism how Gpnmb contributes to neuroprotection is not fully elucidated, but it is suggested that Gpnmb secretion by astrocytes might activate MEK/ERK and PI3K/Akt pathways promoting neuron survival 29. In line with their findings other reports showed that shed Gpnmb promotes cell survival in breast cancer and NIH-3T3 cells 30,31. Katayama et al. showed that Gpnmb derived from Kupffer cells and stellate cells plays a protective role in nonalcoholic fatty liver disease (NAFLD) 32. They show that soluble Gpnmb interacts with calnexin, resulting in a reduction of oxidative stress 32. Our results showed that Gpnmb is highly induced in Kupffer cells in liver and in splenic macrophages of NPC mice (chapter 3). Interestingly, Gpnmb has been shows to play a neuroprotective role in amyotrophic lateral sclerosis as well, suggesting that peripheral Gpnmb expression might be involved in cell survival 29,33. Possibly, Gpnmb as found in our studies contributes to survival as well, but further investigation is required in order to unravel the function of Gpnmb in peripheral tissue in the context of NPC and GD.

Gpnmb has been shown to be involved in innate and adaptive immunity. On one hand Gpnmb binds to dermatophytic fungi potentiating antigen presenting cell function in APC 34. On the other hand, it has been shown that Gpnmb delivers a potent antigen-

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independent inhibitory signal to T cells, by interacting with syndecan-4 (SD-4), a heparin-like saccharide 34,35. Additional analysis in obese adipose tissue should be performed whether SD-4 is present in the adipose tissue and whether Gpnmb is interacting with SD-4 and if this interaction impact in obese conditions.

Another Gpnmb function described is its involvement in osteoclast biology 36,37. Mutated Gpnmb promoted RANKL-mediated osteoclast differentiation and survival but non manipulated Gpnmb inhibited osteoclast function 37. Moreover in another study it was described that Gpnmb interacts with integrin αVβ3 and heparin sulfate proteoglycan (HSPG) promoting osteoclast differentiation 36. All together the data underscore the importance of Gpnmb in osteoclast and bone homeostasis. It is unclear whether Gpnmb is involved in ATM differentiation. In vitro it was shown in RAW264.7 cells that Gpnmb potentiates alternative macrophage activation, suggesting an involvement of Gpnmb in immunosuppression and in macrophage M1/M2 differentiation (chapter 5). In line, recently it has been described that Gpnmb is expressed to a higher level in M2 than M1 bone marrow-derived macrophages 38. Furthermore, in the same study it is shown that secreted Gpnmb from M2 macrophages mediated mesenchymal stem cells (MSCs) proliferation and migration in a CD-44-dependent fashion, promoting a M2 phenotype and tissue repair38. Gpnmb has been described to be involved in tissue repair of ischemic damage in kidney, promoting macroautophagic degradation and phagocytosis, suggesting again its involvement in M2 macrophage function 39. Still, further in vivo experiments are necessary to confirm that Gpnmb contibutes to alternative macrophage activation.

Another possible function of Gpnmb could be more related to its tertiary structure. Gpnmb has a similar structure as Galectin-3 (Gal-3). Gal-3, is induced during inflammation in the AT during obesity, and is highly glycosylated 40–42. Furthermore Gal-3 colocalizes with the endosomal LC3 and is a multifunctional protein involved in many processes, amongst others membrane repair by binding to extracellular carbohydrates structures like muscins and preventing LMP 28,39,43–46. Due to comparable structure, expression pattern and localization with Gal-3, it would be interesting to explore whether Gpnmb plays a similar role in membrane repair and LMP prevention.

Overall many different aspects of Gpnmb, especially its function during obesity in adipose tissue is not fully clear. Katamaya et al. showed that in transgenic mice over expressing Gpnmb fat accumulation and fibrosis of the liver in diet-induced obesity improved 32. Further investigation need to be done in order to elucidate whether

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Gpnmb is involved in membrane repair and macrophage polarization.

Sphingolipid role in cell homeostasis: Sphk1 induction in obese macrophage and the role of GSL iNK function

Analysis of the GSL-metabolizing enzymes during obesity showed a striking induction of Sphk1 in obese ATM (Chapter 6)47. Interestingly, multiple reports showed a systemic increase of SL levels during obesity. In our analysis in obese ATM we showed induction of several (G)SL including GB4, lactosylceramide, sphingosines and sphinganines (Chapter 6). Using the macrophage-like cell line RAW264.7, we showed that Sphk1 induction in obese ATM promotes cell survival when the cells are exposed to palmitate. Interestingly the observed pattern of sphk1 gene expression coincided to some extend with Gpnmb induction (Chapter 6). Palmitate, chloroquine, U18666A, torin 1 and the lysosomal ATPase inhibitor bafilomycin A1 induced both Sphk1 and Gpnmb (data not shown). The magnitude of Sphk1 Gpnmb was not always on a par. Furthermore HEPES that strongly induced Gpnmb failed to induce Sphk1. Inversely, LPS highly induces Sphk1 but not Gpnmp. Combining these findings suggests that the lysosome-to-nucleus signaling axis might also regulate sphk1 to some extent, but the exact mechanism remains to be elucidated. It has been shown that palmitate induction of Sphk1 is peroxisome proliferator-activated receptor α (PPARα) dependent in myocytes 48. PPARα is also expressed in human macrophages, it would be interesting to investigate whether PPARα activation occurs during lysosomal stress and whether PPARα-activation is regulated by mTOR or the MiT family 49.

Several reports showed that S1P promotes cell survival through activation of receptor S1P1

50,51. In this thesis we show that Sphk1 activity and S1P protects RAW264.7 from palmitate induced lipotoxicity (Chapter 6). In agreement, Sphk1 gene and activity levels were increased in obese ATM (Chapter 6). Our studies demonstrated that S1P levels were unchanged when lean ATM were compared with obese ATM (chapter 6). S1P supposedly has three possible fates. Firstly, the S1P generated from the macrophages can be recycled, it can be dephosphorylated to generate sphingosine again and re-enter the ceramide and glycosphingolipid synthesis. S1P can act as a direct intracellular signaling molecule. Alternatively, S1P can be secreted to the extracellular matrix, and act as an inside out signaling promoting S1P receptor (S1PR) activation. Five different S1PR have been described and it has been demonstrated that S1PR1 is involved in promoting cell survival 52. Finally, another possibility could be that S1P generated is quickly degraded allowing to reduce the sphingosine and ceramide levels increased due

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to FFA exposure. Sphingosine and ceramide promote cell death, and their reduction by the generation of S1P and its further catabolized via β-fatty acid oxidation, might contribute to the reduction of the levels of those sphingolipids and in turn promoting cell survival 53. Additional experiments are required in order to elucidate the fate of the S1P generate in obese ATM.

Besides S1P, other SL, including glycosphingolipids, play an important role in lean white adipose tissue homeostasis. In this thesis we show that the enzyme that generates the first building block for more complex sphingolipids, glucosylceramide synthase, through generation of a GSL promotes iNKT cell activation (Chapter 7). The observation that iNKT cells strongly correlate with M2 macrophage as described in other reports, suggests that sphingolipid regulation through lysosomal activity plays an important role in cellular homeostasis during lipid perturbations occurring in GD, NPC and obesity 54.

Future perspectives

This thesis describes Gpnmb as a novel macrophage marker reflecting lysosomal stress caused by improper lipid handling occurring during obesity (Chapter 5) and LSD (Chapter 2 and chapter 3). Gpnmb is highly expressed in macrophages and is MITF and TFEB dependent. A deeper understanding of MiT-dependent Gpnmb regulation is necessary, since MITF and TFEB exist in multiple isoforms. It remains to be determined what MiT homo- and heterodimers exist and if those interactions regulate different sets of genes in a specific fashion (Chapter 4 and Chapter 5). We showed in Chapter 4 that HEPES-induced Gpnmb occurs independent of mTORC1. In line it recently has been described in other systems, including macrophages, that TFEB activation can be mTOR-independent 55–57. It still remains to be elucidated by what mechanism this mTOR-independent MiT activation is caused.

Regarding the function of Gpnmb we showed that Gpnmb potentiates alternative macrophage activation (Chapter 5). Still the mechanism underlying this phenomenon remains to be solved. In line with our immune suppressive finding, other studies showed that Gpnmb reduces T cell responses 34,35. Altogether it seems Gpnmb exerts an anti-inflammatory function, presumably involved in a negative feedback loop preventing an exaggerated immune response. A further investigation is required to mechanistically describe Gpnmb function during obesity.

During obesity and in sphingolipidoses like GD and NPC, lipid accumulation occurs, driving macrophage alterations (Chapter 2, chapter 3 and chapter 5). The research

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presented in this thesis supports the idea that Sphk1 upregulation in obese adipose tissue macrophages protects macrophages from the lipotoxic environment. Numerous studies showed an increase in macrophage number during obesity. It has been suggested that those macrophages contribute to the inflammation and worsening the insulin resistance 58,59. However, recently it has been shown that IL-6 promotes macrophage alternative activation, counteracting obesity-associated insulin resistance suggesting that not all cytokines released by macrophages are contributing to the worsening of inflammation in obesity60. Additionally a study performed by Kraakman et al. showed that it was possible to uncouple macrophage recruitment from insulin resistance, demonstrating that macrophage recruitment is not per se involved in insulin resistance during obesity 61. It has been shown that the macrophage inflammatory response is independent of Sphk1 and in this thesis we show that Sphk1 is essential for macrophage survival under lysosomal stress conditions, induced by excessive lipid load derived from adipocytes, suggesting that ATM might contribute to tissue repair and lipid buffering from malfunctioning adipocytes rather than merely contributing to a pro-inflammatoy state (Chapter 6) 62.

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General discussion

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