Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

S-Nitrosylation of cathepsin B affects autophagic flux and accumulation of protein aggregates in neurodegenerative disorders

Abstract

Protein S-nitrosylation is known to regulate enzymatic function. Here, we report that nitric oxide (NO)-related species can contribute to Alzheimer’s disease (AD) by S-nitrosylating the lysosomal protease cathepsin B (forming SNO-CTSB), thereby inhibiting CTSB activity. This posttranslational modification inhibited autophagic flux, increased autolysosomal vesicles, and led to accumulation of protein aggregates. CA-074Me, a CTSB chemical inhibitor, also inhibited autophagic flux and resulted in accumulation of protein aggregates similar to the effect of SNO-CTSB. Inhibition of CTSB activity also induced caspase-dependent neuronal apoptosis in mouse cerebrocortical cultures. To examine which cysteine residue(s) in CTSB are S-nitrosylated, we mutated candidate cysteines and found that three cysteines were susceptible to S-nitrosylation. Finally, we observed an increase in SNO-CTSB in both 5XFAD transgenic mouse and flash-frozen postmortem human AD brains. These results suggest that S-nitrosylation of CTSB inhibits enzymatic activity, blocks autophagic flux, and thus contributes to AD pathogenesis.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: RNS suppresses autophagic flux.
Fig. 2: RNS augment protein aggregates.
Fig. 3: SNOC regulates cathepsin B activity by S-nitrosylation.
Fig. 4: The CTSB inhibitor, CA-074Me, inhibits autophagic flux mimicking the effect of RNS.
Fig. 5: CTSB inhibition induces neuronal cell apoptosis in mouse cerebrocortical cultures.
Fig. 6: S-Nitrosylated cysteine residues of CTSB.
Fig. 7: S-Nitrosylation and enzymatic inhibition of CTSB in 5X FAD mouse and human AD brains.

Data availability

Correspondence and requests for materials should be addressed to YHK or SAL.

References

  1. de Vrij FM, Fischer DF, van Leeuwen FW, Hol EM. Protein quality control in Alzheimer’s disease by the ubiquitin proteasome system. Prog Neurobiol. 2004;74:249–70.

    Article  PubMed  Google Scholar 

  2. Rubinsztein DC. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 2006;443:780–6.

    Article  CAS  PubMed  Google Scholar 

  3. Upadhya SC, Hegde AN. Ubiquitin-proteasome pathway components as therapeutic targets for CNS maladies. Curr Pharm Des. 2005;11:3807–28.

    Article  CAS  PubMed  Google Scholar 

  4. Barmada SJ, Serio A, Arjun A, Bilican B, Daub A, Ando DM, et al. Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models. Nat Chem Biol. 2014;10:677–85.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, et al. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell 2010;141:1146–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet. 2002;11:1107–17.

    Article  CAS  PubMed  Google Scholar 

  7. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. α-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem. 2003;278:25009–13.

    Article  CAS  PubMed  Google Scholar 

  8. Nandi D, Tahiliani P, Kumar A, Chandu D. The ubiquitin-proteasome system. J Biosci. 2006;31:137–55.

    Article  CAS  PubMed  Google Scholar 

  9. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.

    Article  CAS  PubMed  Google Scholar 

  10. Pohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science 2019;366:818–22.

    Article  CAS  PubMed  Google Scholar 

  11. Juenemann K, Schipper-Krom S, Wiemhoefer A, Kloss A, Sanz Sanz A, Reits EA. Expanded polyglutamine-containing N-terminal huntingtin fragments are entirely degraded by mammalian proteasomes. J Biol Chem. 2013;288:27068–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006;441:885–9.

    Article  CAS  PubMed  Google Scholar 

  13. Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 2006;441:880–4.

    Article  CAS  PubMed  Google Scholar 

  14. Walkley SU, Suzuki K. Consequences of NPC1 and NPC2 loss of function in mammalian neurons. Biochim Biophys Acta. 2004;1685:48–62.

    Article  CAS  PubMed  Google Scholar 

  15. Tessitore A, del PMM, Sano R, Ma Y, Mann L, Ingrassia A, et al. GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis. Mol Cell. 2004;15:753–66.

    Article  CAS  PubMed  Google Scholar 

  16. Sleat DE, Wiseman JA, El-Banna M, Kim KH, Mao Q, Price S, et al. A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration. J Neurosci. 2004;24:9117–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Anda FC, Madabhushi R, Rei D, Meng J, Graff J, Durak O, et al. Cortical neurons gradually attain a post-mitotic state. Cell Res. 2016;26:1033–47.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Chabrier PE, Demerle-Pallardy C, Auguet M. Nitric oxide synthases: targets for therapeutic strategies in neurological diseases. Cell Mol Life Sci. 1999;55:1029–35.

    Article  CAS  PubMed  Google Scholar 

  19. Torreilles F, Salman-Tabcheh S, Guerin M, Torreilles J. Neurodegenerative disorders: the role of peroxynitrite. Brain Res Brain Res Rev. 1999;30:153–63.

    Article  CAS  PubMed  Google Scholar 

  20. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F, Leighton F, et al. The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer’s disease. Prog Neurobiol. 2000;62:633–48.

    Article  CAS  PubMed  Google Scholar 

  21. Simic G, Lucassen PJ, Krsnik Z, Kruslin B, Kostovic I, Winblad B, et al. nNOS expression in reactive astrocytes correlates with increased cell death related DNA damage in the hippocampus and entorhinal cortex in Alzheimer’s disease. Exp Neurol. 2000;165:12–26.

    Article  CAS  PubMed  Google Scholar 

  22. LaVoie MJ, Hastings TG. Peroxynitrite- and nitrite-induced oxidation of dopamine: implications for nitric oxide in dopaminergic cell loss. J Neurochem. 1999;73:2546–54.

    Article  CAS  PubMed  Google Scholar 

  23. Smith BC, Marletta MA. Mechanisms of S-nitrosothiol formation and selectivity in nitric oxide signaling. Curr Opin Chem Biol. 2012;16:498–506.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ohkubo Y, Nakato R, Uehara T. Regulation of unfolded protein response via protein S-nitrosylation. Yakugaku Zasshi. 2016;136:801–4.

    Article  CAS  PubMed  Google Scholar 

  25. Nakato R, Ohkubo Y, Konishi A, Shibata M, Kaneko Y, Iwawaki T, et al. Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress. Sci Rep. 2015;5:14812.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kumar S, Barthwal MK, Dikshit M. Cdk2 nitrosylation and loss of mitochondrial potential mediate NO-dependent biphasic effect on HL-60 cell cycle. Free Radic Biol Med. 2010;48:851–61.

    Article  CAS  PubMed  Google Scholar 

  27. Choi YB, Tenneti L, Le DA, Ortiz J, Bai G, Chen HS, et al. Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation. Nat Neurosci. 2000;3:15–21.

    Article  CAS  PubMed  Google Scholar 

  28. Umanah GKE, Ghasemi M, Yin X, Chang M, Kim JW, Zhang J, et al. AMPA receptor surface expression is regulated by S-nitrosylation of thorase and transnitrosylation of NSF. Cell Rep. 2020;33:108329.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Melino G, Bernassola F, Knight RA, Corasaniti MT, Nistico G, Finazzi-Agro A. S-nitrosylation regulates apoptosis. Nature 1997;388:432–3.

    Article  CAS  PubMed  Google Scholar 

  30. Zhu L, Zhang C, Liu Q. PTEN S-nitrosylation by NOS1 inhibits autophagy in NPC cells. Cell Death Dis. 2019;10:306.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Sarkar S, Korolchuk VI, Renna M, Imarisio S, Fleming A, Williams A, et al. Complex inhibitory effects of nitric oxide on autophagy. Mol Cell. 2011;43:19–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Darios F, Stevanin G. Impairment of lysosome function and autophagy in rare neurodegenerative diseases. J Mol Biol. 2020;432:2714–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shacka JJ, Roth KA, Zhang J. The autophagy-lysosomal degradation pathway: role in neurodegenerative disease and therapy. Front Biosci. 2008;13:718–36.

    Article  CAS  PubMed  Google Scholar 

  34. Kim YH, Kim EY, Gwag BJ, Sohn S, Koh JY. Zinc-induced cortical neuronal death with features of apoptosis and necrosis: mediation by free radicals. Neuroscience 1999;89:175–82.

    Article  CAS  PubMed  Google Scholar 

  35. Lei SZ, Pan ZH, Aggarwal SK, Chen HS, Hartman J, Sucher NJ, et al. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex. Neuron 1992;8:1087–99.

    Article  CAS  PubMed  Google Scholar 

  36. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA. 1995;92:7162–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Koh JY, Choi DW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods. 1987;20:83–90.

    Article  CAS  PubMed  Google Scholar 

  38. Nakamura T, Oh CK, Liao L, Zhang X, Lopez KM, Gibbs D, et al. Noncanonical transnitrosylation network contributes to synapse loss in Alzheimer’s disease. Science 2021;371:eaaw0843.

    Article  CAS  PubMed  Google Scholar 

  39. Oh CK, Sultan A, Platzer J, Dolatabadi N, Soldner F, McClatchy DB, et al. S-Nitrosylation of PINK1 attenuates PINK1/parkin-dependent mitophagy in hiPSC-based Parkinson’s disease models. Cell Rep. 2017;21:2171–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Uehara T, Nakamura T, Yao D, Shi ZQ, Gu Z, Ma Y, et al. S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 2006;441:513–7.

    Article  CAS  PubMed  Google Scholar 

  41. Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol. 2001;3:193–7.

    Article  CAS  PubMed  Google Scholar 

  42. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell 2010;140:313–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Percival MD, Ouellet M, Campagnolo C, Claveau D, Li C. Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid. Biochemistry 1999;38:13574–83.

    Article  CAS  PubMed  Google Scholar 

  44. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, et al. S-Nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci USA. 1992;89:444–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, et al. Exposure of sulphydryl-containing proteins to nitric oxide and endothelium-derived relaxing factor confers novel bioactivity and modulates their intrinsic functional properties. In: Moncada S, Marletta MA, J. B. Hibbs J, Higgs EA, editors. The Biology of Nitric Oxide. London: Portland Press; 1992. p. 20–23.

  46. Zhou Y, Wynia-Smith SL, Couvertier SM, Kalous KS, Marletta MA, Smith BC, et al. Chemoproteomic strategy to quantitatively monitor transnitrosation uncovers functionally relevant S-nitrosations sites on cathepsin D and HADH2. Cell Chem Biol. 2016;23:727–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Gotzl JK, Colombo AV, Fellerer K, Reifschneider A, Werner G, Tahirovic S, et al. Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice. Mol Neurodegener. 2018;13:48.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Pislar A, Kos J. Cysteine cathepsins in neurological disorders. Mol Neurobiol. 2014;49:1017–30.

    Article  CAS  PubMed  Google Scholar 

  49. Bhoopathi P, Chetty C, Gujrati M, Dinh DH, Rao JS, Lakka S. Cathepsin B facilitates autophagy-mediated apoptosis in SPARC overexpressed primitive neuroectodermal tumor cells. Cell Death Differ. 2010;17:1529–39.

    Article  CAS  PubMed  Google Scholar 

  50. Vancompernolle K, Van Herreweghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, et al. Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBS Lett. 1998;438:150–8.

    Article  CAS  PubMed  Google Scholar 

  51. Balboula AZ, Yamanaka K, Sakatani M, Kawahara M, Hegab AO, Zaabel SM, et al. Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus-oocyte complexes exposed to heat shock during in vitro maturation. Reproduction 2013;146:407–17.

    Article  CAS  PubMed  Google Scholar 

  52. Hook V, Toneff T, Bogyo M, Greenbaum D, Medzihradszky KF, Neveu J, et al. Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer’s disease. Biol Chem. 2005;386:931–40.

    Article  CAS  PubMed  Google Scholar 

  53. Kindy MS, Yu J, Zhu H, El-Amouri SS, Hook V, Hook GR. Deletion of the cathepsin B gene improves memory deficits in a transgenic ALZHeimer’s disease mouse model expressing AβPP containing the wild-type β-secretase site sequence. J Alzheimers Dis. 2012;29:827–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hook G, Yu J, Toneff T, Kindy M, Hook V. Brain pyroglutamate amyloid-β is produced by cathepsin B and is reduced by the cysteine protease inhibitor E64d, representing a potential Alzheimer’s disease therapeutic. J Alzheimers Dis. 2014;41:129–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Annis RP, Swahari V, Nakamura A, Xie AX, Hammond SM, Deshmukh M. Mature neurons dynamically restrict apoptosis via redundant premitochondrial brakes. FEBS J. 2016;283:4569–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Putcha GV, Moulder KL, Golden JP, Bouillet P, Adams JA, Strasser A, et al. Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis. Neuron 2001;29:615–28.

    Article  CAS  PubMed  Google Scholar 

  57. Lee JM, Kim YJ, Ra H, Kang SJ, Han S, Koh JY, et al. The involvement of caspase-11 in TPEN-induced apoptosis. FEBS Lett. 2008;582:1871–6.

    Article  CAS  PubMed  Google Scholar 

  58. Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, et al. S-Nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 2002;297:1186–90.

    Article  CAS  PubMed  Google Scholar 

  59. D’Emilia DM, Lipton SA. Ratio of S-nitrosohomocyst(e)ine to homocyst(e)ine or other thiols determines neurotoxicity in rat cerebrocortical cultures. Neurosci Lett. 1999;265:103–6.

    Article  PubMed  Google Scholar 

  60. Kashii S, Mandai M, Kikuchi M, Honda Y, Tamura Y, Kaneda K, et al. Dual actions of nitric oxide in N-methyl-d-aspartate receptor-mediated neurotoxicity in cultured retinal neurons. Brain Res. 1996;711:93–101.

    Article  CAS  PubMed  Google Scholar 

  61. Shimohama S, Akaike A, Kimura J. Nicotine-induced protection against glutamate cytotoxicity. Nicotinic cholinergic receptor-mediated inhibition of nitric oxide formation. Ann NY Acad Sci. 1996;777:356–61.

    Article  CAS  PubMed  Google Scholar 

  62. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 1993;364:626–32.

    Article  CAS  PubMed  Google Scholar 

  63. Chan SJ, San Segundo B, McCormick MB, Steiner DF. Nucleotide and predicted amino acid sequences of cloned human and mouse preprocathepsin B cDNAs. Proc Natl Acad Sci USA. 1986;83:7721–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Fong D, Calhoun DH, Hsieh WT, Lee B, Wells RD. Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B. Proc Natl Acad Sci USA. 1986;83:2909–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, et al. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991;10:2321–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Chagas JR, Ferrer-Di Martino M, Gauthier F, Lalmanach G. Inhibition of cathepsin B by its propeptide: use of overlapping peptides to identify a critical segment. FEBS Lett. 1996;392:233–6.

    Article  CAS  PubMed  Google Scholar 

  67. Song J, Xu P, Xiang H, Su Z, Storer AC, Ni F. The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B. FEBS Lett. 2000;475:157–62.

    Article  CAS  PubMed  Google Scholar 

  68. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007;3:542–5.

    Article  CAS  PubMed  Google Scholar 

  69. Oh CK, Dolatabadi N, Cieplak P, Diaz-Meco MT, Moscat J, Nolan JP, et al. S-Nitrosylation of p62 inhibits autophagic flux to promote α-synuclein secretion and spread in Parkinson’s disease and Lewy body dementia. J Neurosci. 14:JN-RM-1508-21. https://doi.org/10.1523/JNEUROSCI.1508-21.2022.

  70. Oberle C, Huai J, Reinheckel T, Tacke M, Rassner M, Ekert PG, et al. Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes. Cell Death Differ. 2010;17:1167–78.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Eliezer Masliah (UC San Diego/NIA) for providing human brain tissues. This work was supported in part by NIH grants R35 AG071734, R01 NS086890, R01 DA048882, DP1 DA041722, RF1 AG057409, and R01 AG056259 (to SAL), R01 AG061845, RF1 NS123298, and R61 NS122098 (to TN), and National Research Foundation of Korea (NRF) grants NRF-2017M3C7A1028945, NRF-2018R1D1A1B07049746, and NRF-2021R1A2C2008234 (to YHK).

Author information

Authors and Affiliations

Authors

Contributions

YHK and SAL conceived the project and designed the experiments. KRK, EJC, JWE, SSO, TN, and CKO performed experiments. KRK, TN, CKO, SAL and YHK analyzed and interpreted results. KRK, TN, CKO, YHK, and SAL wrote the manuscript.

Corresponding authors

Correspondence to Stuart A. Lipton or Yang-Hee Kim.

Ethics declarations

Competing interests

The authors declare that YHK is a shareholder of Zincure Corp., and that KRK is currently employed by Zincure Corp. SAL is a scientific founder of Adamas Pharmaceuticals, Inc., EuMentis Therapeutics, Inc., and InflaMED Therapeutics, LLC.

Ethical statement and consent to participate

Our studies did not include human participants or human data. Archived human brain samples were analyzed with institutional permission under the state of California and NIH guidelines. Informed consent was obtained according to procedures approved by Institutional Review Boards at the University of California, San Diego, School of Medicine, and The Scripps Research Institute. All animal experimental procedures were approved by the Animal Care and Use Committee of Sejong University and were conducted following the guidelines of the Care and Use of Laboratory Animals.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by G Melino

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kim, KR., Cho, EJ., Eom, JW. et al. S-Nitrosylation of cathepsin B affects autophagic flux and accumulation of protein aggregates in neurodegenerative disorders. Cell Death Differ 29, 2137–2150 (2022). https://doi.org/10.1038/s41418-022-01004-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41418-022-01004-0

Search

Quick links