Abstract
Aging is the largest risk factor for Alzheimer’s disease (AD). Patients with Down syndrome (DS) develop symptoms consistent with early-onset AD, suggesting that overexpression of chromosome 21 genes such as Regulator of Calcineurin 1 (RCAN1) plays a role in AD pathogenesis. RCAN1 levels are increased in the brain of DS and AD patients but also in the human brain with normal aging. RCAN1 has been implicated in several neuronal functions, but whether its increased expression is correlative or causal in the aging-related progression of AD remains elusive. We show that brain-specific overexpression of the human RCAN1.1S isoform in mice promotes early age-dependent memory and synaptic plasticity deficits, tau pathology, and dysregulation of dynamin-related protein 1 (DRP1) activity associated with mitochondrial dysfunction and oxidative stress, reproducing key AD features. Based on these findings, we propose that chronic RCAN1 overexpression during aging alters DRP1-mediated mitochondrial fission and thus acts to promote AD-related progressive neurodegeneration.
Similar content being viewed by others
References
Barria A, Muller D, Derkach V, Griffith LC, Soderling TR (1997) Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. Science 276:2042–2045
Bhoiwala DL, Koleilat I, Qian J, Beyer B, Hushmendy SF, Mathew A, Bhoiwala DL, Ferland RJ, Crawford DR (2013) Overexpression of RCAN1 isoform 4 in mouse neurons leads to a moderate behavioral impairment. Neurol Res 35:79–89. doi:10.1179/1743132812Y.0000000117
Bishop NA, Lu T, Yankner BA (2010) Neural mechanisms of ageing and cognitive decline. Nature 464:529–535. doi:10.1038/nature08983
Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat Rev Neurosci 7:30–40. doi:10.1038/nrn1809
Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, Scorrano L (2008) Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc Natl Acad Sci USA 105:15803–15808. doi:10.1073/pnas.0808249105
Chang KT, Min KT (2005) Drosophila melanogaster homolog of Down syndrome critical region 1 is critical for mitochondrial function. Nat Neurosci 8:1577–1585. doi:10.1038/nn1564
Cook CN, Hejna MJ, Magnuson DJ, Lee JM (2005) Expression of calcipressin1, an inhibitor of the phosphatase calcineurin, is altered with aging and Alzheimer’s disease. J Alzheimers Dis JAD 8:63–73
Cribbs JT, Strack S (2007) Reversible phosphorylation of Drp1 by cyclic AMP-dependent protein kinase and calcineurin regulates mitochondrial fission and cell death. EMBO Rep 8:939–944. doi:10.1038/sj.embor.7401062
Dierssen M, Arque G, McDonald J, Andreu N, Martinez-Cue C, Florez J, Fillat C (2011) Behavioral characterization of a mouse model overexpressing DSCR1/RCAN1. PLoS One 6:e17010. doi:10.1371/journal.pone.0017010
Eckert GP, Renner K, Eckert SH, Eckmann J, Hagl S, Abdel-Kader RM, Kurz C, Leuner K, Muller WE (2012) Mitochondrial dysfunction—a pharmacological target in Alzheimer’s disease. Mol Neurobiol 46:136–150. doi:10.1007/s12035-012-8271-z
English JD, Sweatt JD (1997) A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem 272:19103–19106
Ermak G, Pritchard MA, Dronjak S, Niu B, Davies KJ (2011) Do RCAN1 proteins link chronic stress with neurodegeneration? FASEB J 25:3306–3311. doi:10.1096/fj.11-185728
Ermak G, Sojitra S, Yin F, Cadenas E, Cuervo AM, Davies KJ (2012) Chronic expression of RCAN1-1L protein induces mitochondrial autophagy and metabolic shift from oxidative phosphorylation to glycolysis in neuronal cells. J Biol Chem 287:14088–14098. doi:10.1074/jbc.M111.305342
Giovannini C, Matarrese P, Scazzocchio B, Sanchez M, Masella R, Malorni W (2002) Mitochondria hyperpolarization is an early event in oxidized low-density lipoprotein-induced apoptosis in Caco-2 intestinal cells. FEBS Lett 523:200–206
Gower AJ, Lamberty Y (1993) The aged mouse as a model of cognitive decline with special emphasis on studies in NMRI mice. Behav Brain Res 57:163–173
Harris CD, Ermak G, Davies KJ (2007) RCAN1-1L is overexpressed in neurons of Alzheimer’s disease patients. FEBS J 274:1715–1724. doi:10.1111/j.1742-4658.2007.05717.x
Hebert LE, Weuve J, Scherr PA, Evans DA (2013) Alzheimer disease in the United States (2010-2050) estimated using the 2010 census. Neurology 80:1778–1783. doi:10.1212/WNL.0b013e31828726f5
Hoeffer CA, Dey A, Sachan N, Wong H, Patterson RJ, Shelton JM, Richardson JA, Klann E, Rothermel BA (2007) The Down syndrome critical region protein RCAN1 regulates long-term potentiation and memory via inhibition of phosphatase signaling. J Neurosci 27:13161–13172
Hoeffer CA, Tang W, Wong H, Santillan A, Patterson RJ, Martinez LA, Tejada-Simon MV, Paylor R, Hamilton SL, Klann E (2008) Removal of FKBP12 enhances mTOR-Raptor interactions, LTP, memory, and perseverative/repetitive behavior. Neuron 60:832–845. doi:10.1016/j.neuron.2008.09.037
Hoeffer CA, Wong H, Cain P, Levenga J, Cowansage KK, Choi Y, Davy C, Majmundar N, McMillan DR, Rothermel BA et al (2013) Regulator of calcineurin 1 modulates expression of innate anxiety and anxiogenic responses to selective serotonin reuptake inhibitor treatment. J Neurosci 33:16930–16944. doi:10.1523/JNEUROSCI.3513-12.2013
Horne EA, Dell’Acqua ML (2007) Phospholipase C is required for changes in postsynaptic structure and function associated with NMDA receptor-dependent long-term depression. J Neurosci 27:3523–3534. doi:10.1523/JNEUROSCI.4340-06.2007
Hu D, Serrano F, Oury TD, Klann E (2006) Aging-dependent alterations in synaptic plasticity and memory in mice that overexpress extracellular superoxide dismutase. J Neurosci 26:3933–3941. doi:10.1523/JNEUROSCI.5566-05.2006
Keck S, Nitsch R, Grune T, Ullrich O (2003) Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer’s disease. J Neurochem 85:115–122
Keller JN, Hanni KB, Markesbery WR (2000) Possible involvement of proteasome inhibition in aging: implications for oxidative stress. Mech Ageing Dev 113:61–70
Krinsky-McHale SJ, Devenny DA, Kittler P, Silverman W (2008) Selective attention deficits associated with mild cognitive impairment and early stage Alzheimer’s disease in adults with Down syndrome. Am J Ment Retard AJMR 113:369–386. doi:10.1352/2008.113:369-386
Lemere CA, Masliah E (2010) Can Alzheimer disease be prevented by amyloid-beta immunotherapy? Nat Rev Neurol 6:108–119. doi:10.1038/nrneurol.2009.219
Leuner K, Hauptmann S, Abdel-Kader R, Scherping I, Keil U, Strosznajder JB, Eckert A, Muller WE (2007) Mitochondrial dysfunction: the first domino in brain aging and Alzheimer’s disease? Antioxid Redox Signal 9:1659–1675
Levenga J, Krishnamurthy P, Rajamohamedsait H, Wong H, Franke TF, Cain P, Sigurdsson EM, Hoeffer CA (2013) Tau pathology induces loss of GABAergic interneurons leading to altered synaptic plasticity and behavioral impairments. Acta Neuropathol Commun 1:34. doi:10.1186/2051-5960-1-34
Li Z, Okamoto K, Hayashi Y, Sheng M (2004) The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119:873–887. doi:10.1016/j.cell.2004.11.003
Liu Q, Busby JC, Molkentin JD (2009) Interaction between TAK1-TAB 1-TAB 2 and RCAN1-calcineurin defines a signalling nodal control point. Nat Cell Biol 11:154–161. doi:10.1038/ncb1823
Lloret A, Badia MC, Giraldo E, Ermak G, Alonso MD, Pallardo FV, Davies KJ, Vina J (2011) Amyloid-beta toxicity and tau hyperphosphorylation are linked via RCAN1 in Alzheimer’s disease. J Alzheimers Dis JAD 27:701–709. doi:10.3233/JAD-2011-110890
Lott IT, Head E (2001) Down syndrome and Alzheimer’s disease: a link between development and aging. Ment Retard Dev Disabil Res Rev 7:172–178. doi:10.1002/mrdd.1025
Ma H, Xiong H, Liu T, Zhang L, Godzik A, Zhang Z (2004) Aggregate formation and synaptic abnormality induced by DSCR1. J Neurochem 88:1485–1496
Ma T, Hoeffer CA, Wong H, Massaad CA, Zhou P, Iadecola C, Murphy MP, Pautler RG, Klann E (2011) Amyloid beta-induced impairments in hippocampal synaptic plasticity are rescued by decreasing mitochondrial superoxide. J Neurosci 31:5589–5595. doi:10.1523/JNEUROSCI.6566-10.2011
Maccioni RB, Munoz JP, Barbeito L (2001) The molecular bases of Alzheimer’s disease and other neurodegenerative disorders. Arch Med Res 32:367–381
Manczak M, Calkins MJ, Reddy PH (2011) Impaired mitochondrial dynamics and abnormal interaction of amyloid beta with mitochondrial protein Drp1 in neurons from patients with Alzheimer’s disease: implications for neuronal damage. Hum Mol Genet 20:2495–2509. doi:10.1093/hmg/ddr139
Manczak M, Reddy PH (2012) Abnormal interaction between the mitochondrial fission protein Drp1 and hyperphosphorylated tau in Alzheimer’s disease neurons: implications for mitochondrial dysfunction and neuronal damage. Hum Mol Genet 21:2538–2547
Markesbery WR (1997) Oxidative stress hypothesis in Alzheimer’s disease. Free Radic Biol Med 23:134–147
Martin KR, Corlett A, Dubach D, Mustafa T, Coleman HA, Parkington HC, Merson TD, Bourne JA, Porta S, Arbones ML et al (2012) Over-expression of RCAN1 causes Down syndrome-like hippocampal deficits that alter learning and memory. Hum Mol Genet 21:3025–3041. doi:10.1093/hmg/dds134
Martinez-Martinez S, Genesca L, Rodriguez A, Raya A, Salichs E, Were F, Lopez-Maderuelo MD, Redondo JM, de la Luna S (2009) The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators. Proc Natl Acad Sci USA 106:6117–6122. doi:10.1073/pnas.0812544106
Mawal-Dewan M, Henley J, Van de Voorde A, Trojanowski JQ, Lee VM (1994) The phosphorylation state of tau in the developing rat brain is regulated by phosphoprotein phosphatases. J Biol Chem 269:30981–30987
Nelson PT, Alafuzoff I, Bigio EH, Bouras C, Braak H, Cairns NJ, Castellani RJ, Crain BJ, Davies P, Del Tredici K et al (2012) Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J Neuropathol Exp Neurol 71:362–381. doi:10.1097/NEN.0b013e31825018f7
Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM (2003) Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39:409–421
Okazawa M, Abe H, Katsukawa M, Iijima K, Kiwada T, Nakanishi S (2009) Role of calcineurin signaling in membrane potential-regulated maturation of cerebellar granule cells. J Neurosci 29:2938–2947. doi:10.1523/JNEUROSCI.5932-08.2009
Pater C (2011) Mild cognitive impairment (MCI)—the novel trend of targeting Alzheimer’s disease in its early stages—methodological considerations. Curr Alzheimer Res 8:798–807
Peiris H, Dubach D, Jessup CF, Unterweger P, Raghupathi R, Muyderman H, Zanin MP, Mackenzie K, Pritchard MA, Keating DJ (2014) RCAN1 regulates mitochondrial function and increases susceptibility to oxidative stress in mammalian cells. Oxid Med Cell Longev 2014:520316. doi:10.1155/2014/520316
Perluigi M, Di Domenico F, Buttterfield DA (2014) Unraveling the complexity of neurodegeneration in brains of subjects with Down syndrome: insights from proteomics. Proteomics Clin Appl 8:73–85. doi:10.1002/prca.201300066
Poppek D, Keck S, Ermak G, Jung T, Stolzing A, Ullrich O, Davies KJ, Grune T (2006) Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress. Biochem J 400:511–520. doi:10.1042/BJ20060463
Rainero I, Bergamini L, Bruni AC, Ferini-Strambi L, Foncin JF, Gei G, Macciardi F, Montesi MP, Pinessi L, Vaula G (1994) A new Italian pedigree with early-onset Alzheimer’s disease. J Geriatr Psychiatry Neurol 7:28–32
Reddy PH, Beal MF (2005) Are mitochondria critical in the pathogenesis of Alzheimer’s disease? Brain Res Brain Res Rev 49:618–632. doi:10.1016/j.brainresrev.2005.03.004
Reddy PH, Tripathi R, Troung Q, Tirumala K, Reddy TP, Anekonda V, Shirendeb UP, Calkins MJ, Reddy AP, Mao P et al (2012) Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer’s disease: implications to mitochondria-targeted antioxidant therapeutics. Biochim Biophys Acta 1822:639–649. doi:10.1016/j.bbadis.2011.10.011
Rothermel B, Vega RB, Yang J, Wu H, Bassel-Duby R, Williams RS (2000) A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling. J Biol Chem 275:8719–8725
Shukkur EA, Shimohata A, Akagi T, Yu W, Yamaguchi M, Murayama M, Chui D, Takeuchi T, Amano K, Subramhanya KH et al (2006) Mitochondrial dysfunction and tau hyperphosphorylation in Ts1Cje, a mouse model for Down syndrome. Hum Mol Genet 15:2752–2762. doi:10.1093/hmg/ddl211
Smith RA, Murphy MP (2010) Animal and human studies with the mitochondria-targeted antioxidant MitoQ. Ann N Y Acad Sci 1201:96–103. doi:10.1111/j.1749-6632.2010.05627.x
Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK et al (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8:1051–1058
Sun X, Wu Y, Chen B, Zhang Z, Zhou W, Tong Y, Yuan J, Xia K, Gronemeyer H, Flavell RA et al (2011) Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation. J Biol Chem 286:9049–9062. doi:10.1074/jbc.M110.177519
Tsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87:1317–1326
Wang X, Su B, Lee HG, Li X, Perry G, Smith MA, Zhu X (2009) Impaired balance of mitochondrial fission and fusion in Alzheimer’s disease. J Neurosci 29:9090–9103
Wang X, Su B, Zheng L, Perry G, Smith MA, Zhu X (2009) The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer’s disease. J Neurochem 109(Suppl 1):153–159. doi:10.1111/j.1471-4159.2009.05867.x
Ward MW, Huber HJ, Weisova P, Dussmann H, Nicholls DG, Prehn JH (2007) Mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate-induced necrosis, apoptosis, and tolerance. J Neurosci 27:8238–8249
Wu Y, Song W (2013) Regulation of RCAN1 translation and its role in oxidative stress-induced apoptosis. FASEB J 27:208–221. doi:10.1096/fj.12-213124
Xing L, Salas M, Zhang H, Gittler J, Ludwig T, Lin CS, Murty VV, Silverman W, Arancio O, Tycko B (2013) Creation and characterization of BAC-transgenic mice with physiological overexpression of epitope-tagged RCAN1 (DSCR1). Mamm Genome 24:30–43. doi:10.1007/s00335-012-9436-9
Yang J, Rothermel B, Vega RB, Frey N, McKinsey TA, Olson EN, Bassel-Duby R, Williams RS (2000) Independent signals control expression of the calcineurin inhibitory proteins MCIP1 and MCIP2 in striated muscles. Circ Res 87:E61–E68
Zhou C, Tu J, Zhang Q, Lu D, Zhu Y, Zhang W, Yang F, Brann DW, Wang R (2011) Delayed ischemic postconditioning protects hippocampal CA1 neurons by preserving mitochondrial integrity via Akt/GSK3beta signaling. Neurochem Int 59:749–758
Acknowledgments
For technical assistance, resources, and funding, we thank Yoon Choi, Areum Kang, Pavan Krishnamurthy, Michael Murphy, Chris Link, BBDP tissue bank, CU Boulder BioFrontiers Microscopy Core, Alzheimer’s Association MNIRGDP-12-258900 (CAH), NARSAD 21069 (CAH), NIH F31 NS083277 (HW), NIH T32 MH019524 (HW), Simons Foundation SFARI 27444 (CAH), Sie Foundation (JL).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
401_2015_1499_MOESM1_ESM.pdf
Supplementary material 1: Fig. 1. RCAN1 expression in the brain. (a) Ratio of RCAN1.1 isoforms expressed in the brain of Alzheimer’s disease (AD) patients and age-matched controls (CTRL) were similar (t (21) = .789, p = .439). N = 12 CTRL, 11 AD. (b) Ratio of RCAN1.1 isoforms expressed in the hippocampus of RCAN1 TG mice did not change significantly with age (t (14) = 1.641, p = .123). N = 8 mice/group. (c) mRNA levels of the RCAN1.1S transgene (FLAG-RCAN1.1S) did not change during aging (t (4) = .639, p = .226). N = 3 mice/group. (d) Representative western blots comparing RCAN1 isoform expression in different brain regions of young (3-5 months old) vs. aged (12-14 months old) TG and WT littermates. Olfactory bulb (OLF), prefrontal cortex (PFC), cortex (CTX), hippocampus (HIP), cerebellum (CER), and striatum (STR). (e) Immunohistochemical staining of RCAN1 in hippocampal area CA1 showed increased signal with RCAN1.1S overexpression and with age. Signal specificity confirmed with Rcan1 knockout (KO) hippocampal slices. Hoechst nuclear stain. Scale bar 10 µm. Images representative of 3 independent experimental cohorts, 3 slices/mouse. *p < .05. (PDF 256 kb)
401_2015_1499_MOESM2_ESM.pdf
Supplementary material 2: Fig. 2. Early pathological tau staining in the hippocampus of RCAN1 TG mice. (a) AT8 staining of area CA1 in the hippocampus confirmed our western results showing similarly high levels of early pathological tau phosphorylation in young TG mice and the aged group compared with young WT mice (Fig. 4). Additionally, AT8 staining seemed to show a different localization pattern in TG mice from both age groups compared with WT mice. (b) Images of AT8 co-staining with total tau (left) and background staining from secondary antibodies absent of primary antibodies (right). Images representative of 3 independent experimental cohorts, 3 slices/mouse. Scale bar, 10 µm. (PDF 238 kb)
401_2015_1499_MOESM3_ESM.pdf
Supplementary material 3: Fig. 3. Normal LTP response to mitochondrial challenge in young RCAN1 TG mice. Antimycin (ant) treatment of hippocampal slices impaired E-LTP similarly in young TG and WT mice compared with vehicle (veh) treatment (F (3,88) = 7.383, p < .001; main effect of genotype F (1,88) = .007, p = .935; main effect of treatment F (1,88) = 18.367, p < .0001; main effect of interaction F (1,88) = .093, p = .762). n = 21-25 slices/group, 5-7 mice/genotype. Mean fEPSP slopes recorded during last 20 min post-LTP induction shown at right. (PDF 39 kb)
401_2015_1499_MOESM4_ESM.pdf
Supplementary material 4: Fig. 4. DRP1 regulation in the hippocampus of RCAN1 TG mice. (a) Western blots showing all aged samples used for analysis of S637-phosphorylated DRP1 (p-DRP1) levels in Fig. 7a. Data were pooled from 2 independent experimental cohorts: aged (I) and aged (II), which both showed a reduction of hippocampal p-DRP1 levels in aged TG mice compared with WT littermates. Upper aged (I) blots are extended images of the representative western shown in Fig. 7a. Lower blots show all aged samples from both experimental cohorts loaded on the same gel. Normalized to total DRP1 levels. N = 5 mice/genotype. (b) Western blot analysis of hippocampal lysates showing similar p-DRP1 levels normalized to DRP1 (t (8) = -1.130, p = .211) and total DRP1 levels normalized to GAPDH (t (8) = -.755, p = .472) between young WT and TG mice. N = 4-6 mice/group. (c) DRP1 staining showed similar subcellular localization of DRP1 in young TG and WT littermates, as measured by the median DRP1 pixel intensity value in the hippocampal CA1 stratum pyramidale (t (37) = .792, p = .434) and stratum radiatum (t (37) = .269, p = .790). N = 6-7 mice/group, 3 slices/mouse. Scale bar 5 µm. (PDF 173 kb)
Rights and permissions
About this article
Cite this article
Wong, H., Levenga, J., Cain, P. et al. RCAN1 overexpression promotes age-dependent mitochondrial dysregulation related to neurodegeneration in Alzheimer’s disease. Acta Neuropathol 130, 829–843 (2015). https://doi.org/10.1007/s00401-015-1499-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00401-015-1499-8