Aβ mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease

Abstract

Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aβ load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression. SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

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References 70

1. 1.

   Ahmad F, Singh K, Das D, Gowaikar R, Shaw E, Ramachandran A, Rupanagudi KV, Kommaddi RP, Bennett DA, Ravindranath V (2017 ) Reactive oxygen species-mediated loss of synaptic Akt1 signaling leads to deficient activity-dependent protein translation early in Alzheimer's disease. Antioxid Redox Signal 27:1269–1280.

   Not in our library10.1089/ars.2016.6860

2. 2.

   Bamburg JR (1999 ) Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15:185–230.

   Not in our library10.1146/annurev.cellbio.15.1.185

3. 3.

   Bamburg JR, Bernstein BW (2010 ) Roles of ADF/cofilin in actin polymerization and beyond. F1000 Biol Rep 2:62.

   Not in our library10.3410/b2-62

4. 4.

   Bamburg JR, Bernstein BW (2016 ) Actin dynamics and cofilin-actin rods in alzheimer disease. Cytoskeleton (Hoboken) 73:477–497.

   Not in our library10.1002/cm.21282

5. 5.

   Bamburg JR, Bernstein BW, Davis RC, Flynn KC, Goldsbury C, Jensen JR, Maloney MT, Marsden IT, Minamide LS, Pak CW, Shaw AE, Whiteman I, Wiggan O (2010 ) ADF/Cofilin-actin rods in neurodegenerative diseases. Curr Alzheimer Res 7:241–250.

   Not in our library10.2174/156720510791050902

6. 6.

   Barone E, Mosser S, Fraering PC (2014 ) Inactivation of brain Cofilin-1 by age, Alzheimer's disease and gamma-secretase. Biochim Biophys Acta 1842:2500–2509.

   Not in our library10.1016/j.bbadis.2014.10.004

7. 7.

   Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt LF, Arikkath J (2012 ) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754.

   Not in our library10.1038/nprot.2012.099

8. 8.

   Bennett DA, Wilson RS, Schneider JA, Evans DA, Beckett LA, Aggarwal NT, Barnes LL, Fox JH, Bach J (2002 ) Natural history of mild cognitive impairment in older persons. Neurology 59:198–205.

   Not in our library10.1212/wnl.59.2.198

9. 9.

   Bennett DA, Schneider JA, Wilson RS, Bienias JL, Arnold SE (2004 ) Neurofibrillary tangles mediate the association of amyloid load with clinical Alzheimer disease and level of cognitive function. Arch Neurol 61:378–384.

   Not in our library10.1001/archneur.61.3.378

10. 10.

    Bennett DA, Schneider JA, Bienias JL, Evans DA, Wilson RS (2005 ) Mild cognitive impairment is related to Alzheimer disease pathology and cerebral infarctions. Neurology 64:834–841.

    Not in our library10.1212/01.wnl.0000152982.47274.9e

11. 11.

    Bennett DA, Schneider JA, Aggarwal NT, Arvanitakis Z, Shah RC, Kelly JF, Fox JH, Cochran EJ, Arends D, Treinkman AD, Wilson RS (2006 ) Decision rules guiding the clinical diagnosis of Alzheimer's disease in two community-based cohort studies compared to standard practice in a clinic-based cohort study. Neuroepidemiology 27:169–176.

    Not in our library10.1159/000096129

12. 12.

    Bennett DA, Schneider JA, Arvanitakis Z, Wilson RS (2012 ) Overview and findings from the religious orders study. Curr Alzheimer Res 9:628–645.

    Not in our library10.2174/156720512801322573

13. 13.

    Bernstein BW, Bamburg JR (2010 ) ADF/cofilin: a functional node in cell biology. Trends Cell Biol 20:187–195.

    Not in our library10.1016/j.tcb.2010.01.001

14. 14.

    Blanpied TA, Ehlers MD (2004 ) Microanatomy of dendritic spines: emerging principles of synaptic pathology in psychiatric and neurological disease. Biol Psychiatry 55:1121–1127.

    Not in our library10.1016/j.biopsych.2003.10.006

15. 15.

    Bonardi C, de Pulford F, Jennings D, Pardon MC (2011 ) A detailed analysis of the early context extinction deficits seen in APPswe/PS1dE9 female mice and their relevance to preclinical Alzheimer's disease. Behav Brain Res 222:89–97.

    Not in our library10.1016/j.bbr.2011.03.041

16. 16.

    Bosch M, Castro J, Saneyoshi T, Matsuno H, Sur M, Hayashi Y (2014 ) Structural and molecular remodeling of dendritic spine substructures during long-term potentiation. Neuron 82:444–459.

    Not in our library10.1016/j.neuron.2014.03.021

17. 17.

    Boudaoud A, Burian A, Borowska-Wykręt D, Uyttewaal M, Wrzalik R, Kwiatkowska D, Hamant O (2014 ) FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images. Nat Protoc 9:457–463.

    Not in our library10.1038/nprot.2014.024

18. 18.

    Chazeau A, Mehidi A, Nair D, Gautier JJ, Leduc C, Chamma I, Kage F, Kechkar A, Thoumine O, Rottner K, Choquet D, Gautreau A, Sibarita JB, Giannone G (2014 ) Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion. EMBO J 33:2745–2764.

    Not in our library10.15252/embj.201488837

19. 19.

    Colella AD, Chegenii N, Tea MN, Gibbins IL, Williams KA, Chataway TK (2012 ) Comparison of Stain-Free gels with traditional immunoblot loading control methodology. Anal Biochem 430:108–110.

    Not in our library10.1016/j.ab.2012.08.015

20. 20.

    Counts SE, He B, Nadeem M, Wuu J, Scheff SW, Mufson EJ (2012 ) Hippocampal drebrin loss in mild cognitive impairment. Neurodegener Dis 10:216–219.

    Not in our library10.1159/000333122

21. 21.

    Cummings DM, Liu W, Portelius E, Bayram S, Yasvoina M, Ho SH, Smits H, Ali SS, Steinberg R, Pegasiou CM, James OT, Matarin M, Richardson JC, Zetterberg H, Blennow K, Hardy JA, Salih DA, Edwards FA (2015 ) First effects of rising amyloid-beta in transgenic mouse brain: synaptic transmission and gene expression. Brain 138:1992–2004.

    Not in our library10.1093/brain/awv127

22. 22.

    Dickstein DL, Dickstein DR, Janssen WG, Hof PR, Glaser JR, Rodriguez A, O'Connor N, Angstman P, Tappan SJ (2016 ) Automatic dendritic spine quantification from confocal data with Neurolucida 360. Curr Protoc Neurosci 77:1.27.1–1.27.21.

    Not in our library10.1002/cpns.16

23. 23.

    Dillon C, Goda Y (2005 ) The actin cytoskeleton: integrating form and function at the synapse. Annu Rev Neurosci 28:25–55.

    Not in our library10.1146/annurev.neuro.28.061604.135757

24. 24.

    Du H, Guo L, Yan SS (2012 ) Synaptic mitochondrial pathology in Alzheimer's disease. Antioxid Redox Signal 16:1467–1475.

    Not in our library10.1089/ars.2011.4277

25. 25.

    Efimova N, Korobova F, Stankewich MC, Moberly AH, Stolz DB, Wang J, Kashina A, Ma M, Svitkina T (2017 ) BetaIII spectrin is necessary for formation of the constricted neck of dendritic spines and regulation of synaptic activity in neurons. J Neurosci 37:6442–6459.

    Not in our library10.1523/jneurosci.3520-16.2017

26. 26.

    Franklin KBJ, Paxinos G (1997 ) The mouse brain in stereotaxic coordinates. San Diego, CA: Academic Press.

    Not in our library

27. 27.

    Frost NA, Shroff H, Kong H, Betzig E, Blanpied TA (2010 ) Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. Neuron 67:86–99.

    Not in our library10.1016/j.neuron.2010.05.026

28. 28.

    Gilda JE, Gomes AV (2013 ) Stain-Free total protein staining is a superior loading control to beta-actin for Western blots. Anal Biochem 440:186–188.

    Not in our library10.1016/j.ab.2013.05.027

29. 29.

    Gilda JE, Gomes AV (2015 ) Western blotting using in-gel protein labeling as a normalization control: stain-free technology. Methods Mol Biol 1295:381–391.

    Not in our library10.1007/978-1-4939-2550-6_27

30. 30.

    Gu J, Lee CW, Fan Y, Komlos D, Tang X, Sun C, Yu K, Hartzell HC, Chen G, Bamburg JR, Zheng JQ (2010 ) ADF/cofilin-mediated actin dynamics regulate AMPA receptor trafficking during synaptic plasticity. Nat Neurosci 13:1208–1215.

    Not in our library10.1038/nn.2634

31. 31.

    Gürtler A, Kunz N, Gomolka M, Hornhardt S, Friedl AA, McDonald K, Kohn JE, Posch A (2013 ) Stain-Free technology as a normalization tool in Western blot analysis. Anal Biochem 433:105–111.

    Not in our library10.1016/j.ab.2012.10.010

32. 32.

    Heredia L, Helguera P, de Olmos S, Kedikian G, Solá Vigo F, LaFerla F, Staufenbiel M, de Olmos J, Busciglio J, Cáceres A, Lorenzo A (2006 ) Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease. J Neurosci 26:6533–6542.

    Not in our library10.1523/jneurosci.5567-05.2006

33. 33.

    Herms J, Dorostkar MM (2016 ) Dendritic spine pathology in neurodegenerative diseases. Annu Rev Pathol 11:221–250.

    Not in our library10.1146/annurev-pathol-012615-044216

34. 34.

    Holcomb L, Gordon MN, McGowan E, Yu X, Benkovic S, Jantzen P, Wright K, Saad I, Mueller R, Morgan D, Sanders S, Zehr C, O'Campo K, Hardy J, Prada CM, Eckman C, Younkin S, Hsiao K, Duff K (1998 ) Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat Med 4:97–100.

    Not in our library10.1038/nm0198-097

35. 35.

    Hotulainen P, Hoogenraad CC (2010 ) Actin in dendritic spines: connecting dynamics to function. J Cell Biol 189:619–629.

    Not in our library10.1083/jcb.201003008

36. 36.

    Hotulainen P, Paunola E, Vartiainen MK, Lappalainen P (2005 ) Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells. Mol Biol Cell 16:649–664.

    Not in our library10.1091/mbc.e04-07-0555

37. 37.

    Hotulainen P, Llano O, Smirnov S, Tanhuanpää K, Faix J, Rivera C, Lappalainen P (2009 ) Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J Cell Biol 185:323–339.

    Not in our library10.1083/jcb.200809046

38. 38.

    Huang W, Zhu PJ, Zhang S, Zhou H, Stoica L, Galiano M, Krnjević K, Roman G, Costa-Mattioli M (2013 ) mTORC2 controls actin polymerization required for consolidation of long-term memory. Nat Neurosci 16:441–448.

    Not in our library10.1038/nn.3351

39. 39.

    Hung RJ, Spaeth CS, Yesilyurt HG, Terman JR (2013 ) SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics. Nat Cell Biol 15:1445–1454.

    Not in our library10.1038/ncb2871

40. 40.

    Izeddin I, Boulanger J, Racine V, Specht CG, Kechkar A, Nair D, Triller A, Choquet D, Dahan M, Sibarita JB (2012 ) Wavelet analysis for single molecule localization microscopy. Opt Express 20:2081–2095.

    Not in our library10.1364/oe.20.002081

41. 41.

    Jack CR Jr, Holtzman DM (2013 ) Biomarker modeling of Alzheimer's disease. Neuron 80:1347–1358.

    Not in our library10.1016/j.neuron.2013.12.003

42. 42.

    Jack CR Jr, Knopman DS, Jagust WJ, Petersen RC, Weiner MW, Aisen PS, Shaw LM, Vemuri P, Wiste HJ, Weigand SD, Lesnick TG, Pankratz VS, Donohue MC, Trojanowski JQ (2013 ) Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers. Lancet Neurol 12:207–216.

    Not in our library10.1016/s1474-4422(12)70291-0

43. 43.

    Kilgore M, Miller CA, Fass DM, Hennig KM, Haggarty SJ, Sweatt JD, Rumbaugh G (2010 ) Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer's disease. Neuropsychopharmacology 35:870–880.

    Not in our library10.1038/npp.2009.197

44. 44.

    Korobova F, Svitkina T (2010 ) Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis. Mol Biol Cell 21:165–176.

    Not in our library10.1091/mbc.e09-07-0596

45. 45.

    Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH (2006 ) A specific amyloid-beta protein assembly in the brain impairs memory. Nature 440:352–357.

    Not in our library10.1038/nature04533

46. 46.

    Liu DS, Pan XD, Zhang J, Shen H, Collins NC, Cole AM, Koster KP, Ben Aissa M, Dai XM, Zhou M, Tai LM, Zhu YG, LaDu M, Chen XC (2015 ) APOE4 enhances age-dependent decline in cognitive function by down-regulating an NMDA receptor pathway in EFAD-Tg mice. Mol Neurodegener 10:7.

    Not in our library10.1186/s13024-015-0002-2

47. 47.

    Mendoza-Naranjo A, Contreras-Vallejos E, Henriquez DR, Otth C, Bamburg JR, Maccioni RB, Gonzalez-Billault C (2012 ) Fibrillar amyloid-beta1–42 modifies actin organization affecting the cofilin phosphorylation state: a role for Rac1/cdc42 effector proteins and the slingshot phosphatase. J Alzheimers Dis 29:63–77.

    Not in our library10.3233/jad-2012-101575

48. 48.

    Nair D, Hosy E, Petersen JD, Constals A, Giannone G, Choquet D, Sibarita JB (2013 ) Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J Neurosci 33:13204–13224.

    Not in our library10.1523/jneurosci.2381-12.2013

49. 49.

    Nanguneri S, Flottmann B, Horstmann H, Heilemann M, Kuner T (2012 ) Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples. PLoS One 7:e38098.

    Not in our library10.1371/journal.pone.0038098

50. 50.

    Okamoto K, Nagai T, Miyawaki A, Hayashi Y (2004 ) Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 7:1104–1112.

    Not in our library10.1038/nn1311

51. 51.

    Okamoto K, Narayanan R, Lee SH, Murata K, Hayashi Y (2007 ) The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure. Proc Natl Acad Sci U S A 104:6418–6423.

    Not in our library10.1073/pnas.0701656104

52. 52.

    Rivero-Gutiérrez B, Anzola A, Martínez-Augustin O, de Medina FS (2014 ) Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting. Anal Biochem 467:1–3.

    Not in our library10.1016/j.ab.2014.08.027

53. 53.

    Rust MJ, Bates M, Zhuang X (2006 ) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795.

    Not in our library10.1038/nmeth929

54. 54.

    Saito T, Matsuba Y, Yamazaki N, Hashimoto S, Saido TC (2016 ) Calpain activation in Alzheimer's model mice is an artifact of APP and presenilin overexpression. J Neurosci 36:9933–9936.

    Not in our library10.1523/jneurosci.1907-16.2016

55. 55.

    Shankar GM, Li S, Mehta TH, Garcia-Munoz A, Shepardson NE, Smith I, Brett FM, Farrell MA, Rowan MJ, Lemere CA, Regan CM, Walsh DM, Sabatini BL, Selkoe DJ (2008 ) Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 14:837–842.

    Not in our library10.1038/nm1782

56. 56.

    Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J, Nguyen PT, Bacskai BJ, Hyman BT (2005 ) Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci 25:7278–7287.

    Not in our library10.1523/jneurosci.1879-05.2005

57. 57.

    Spires-Jones TL, Meyer-Luehmann M, Osetek JD, Jones PB, Stern EA, Bacskai BJ, Hyman BT (2007 ) Impaired spine stability underlies plaque-related spine loss in an Alzheimer's disease mouse model. Am J Pathol 171:1304–1311.

    Not in our library10.2353/ajpath.2007.070055

58. 58.

    Star EN, Kwiatkowski DJ, Murthy VN (2002 ) Rapid turnover of actin in dendritic spines and its regulation by activity. Nat Neurosci 5:239–246.

    Not in our library10.1038/nn811

59. 59.

    Steger C (1998 ) An unbiased detector of curvilinear structures. IEEE Trans Pattern Anal 20:113–125.

    Not in our library10.1109/34.659930

60. 60.

    Tang AH, Chen H, Li TP, Metzbower SR, MacGillavry HD, Blanpied TA (2016 ) A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature 536:210–214.

    Not in our library10.1038/nature19058

61. 61.

    Terman JR, Kashina A (2013 ) Post-translational modification and regulation of actin. Curr Opin Cell Biol 25:30–38.

    Not in our library10.1016/j.ceb.2012.10.009

62. 62.

    Tsai J, Grutzendler J, Duff K, Gan WB (2004 ) Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat Neurosci 7:1181–1183.

    Not in our library10.1038/nn1335

63. 63.

    van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011 ) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6:991–1009.

    Not in our library10.1038/nprot.2011.336

64. 64.

    Venkataramani V, Herrmannsdörfer F, Heilemann M, Kuner T (2016 ) SuReSim: simulating localization microscopy experiments from ground truth models. Nat Methods 13:319–321.

    Not in our library10.1038/nmeth.3775

65. 65.

    Volianskis A, Køstner R, Mølgaard M, Hass S, Jensen MS (2010 ) Episodic memory deficits are not related to altered glutamatergic synaptic transmission and plasticity in the CA1 hippocampus of the APPswe/PS1deltaE9-deleted transgenic mice model of ss-amyloidosis. Neurobiol Aging 31:1173–1187.

    Not in our library10.1016/j.neurobiolaging.2008.08.005

66. 66.

    Wilson RS, Beckett LA, Barnes LL, Schneider JA, Bach J, Evans DA, Bennett DA (2002 ) Individual differences in rates of change in cognitive abilities of older persons. Psychol Aging 17:179–193.

    Not in our library10.1037/0882-7974.17.2.179

67. 67.

    Woo JA, Zhao X, Khan H, Penn C, Wang X, Joly-Amado A, Weeber E, Morgan D, Kang DE (2015 ) Slingshot-Cofilin activation mediates mitochondrial and synaptic dysfunction via Abeta ligation to beta1-integrin conformers. Cell Death Differ 22:921–934.

    Not in our library10.1038/cdd.2015.5

68. 68.

    Wu HY, Hudry E, Hashimoto T, Kuchibhotla K, Rozkalne A, Fan Z, Spires-Jones T, Xie H, Arbel-Ornath M, Grosskreutz CL, Bacskai BJ, Hyman BT (2010 ) Amyloid beta induces the morphological neurodegenerative triad of spine loss, dendritic simplification, and neuritic dystrophies through calcineurin activation. J Neurosci 30:2636–2649.

    Not in our library10.1523/jneurosci.4456-09.2010

69. 69.

    Xu K, Zhong G, Zhuang X (2013 ) Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339:452–456.

    Not in our library10.1126/science.1232251

70. 70.

    Zhao L, Ma QL, Calon F, Harris-White ME, Yang F, Lim GP, Morihara T, Ubeda OJ, Ambegaokar S, Hansen JE, Weisbart RH, Teter B, Frautschy SA, Cole GM (2006 ) Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat Neurosci 9:234–242.

    Not in our library10.1038/nn1630

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