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RCAN1 overexpression promotes age-dependent mitochondrial dysregulation related to neurodegeneration in Alzheimer’s disease

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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.

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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).

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Correspondence to Charles Hoeffer.

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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)

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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

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