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OCIAD1 Contributes to Neurodegeneration in Alzheimer's Disease by Inducing Mitochondria Dysfunction, Neuronal Vulnerability and Synaptic Damages

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OCIAD1 Contributes to Neurodegeneration in Alzheimer's Disease by Inducing Mitochondria Dysfunction, Neuronal Vulnerability and Synaptic Damages

Xuping Li et al. EBioMedicine.

Abstract

Background: Hyperamyloidosis in the brain is known as the earliest neuropathological change and a unique etiological factor in Alzheimer's disease (AD), while progressive neurodegeneration in certain vulnerable brain regions forms the basis of clinical syndromes. It is not clear how early hyperamyloidosis is implicated in progressive neurodegeneration and what factors contribute to the selective brain vulnerability in AD.

Methods: Bioinformatics and experimental neurobiology methods were integrated to identify novel factors involved in the hyperamyloidosis-induced brain vulnerability in AD. We first examined neurodegeneration-specific gene signatures from sporadic AD patients and synaptic protein changes in young transgenic AD mice. Then, we systematically assessed the association of a top candidate gene with AD and investigated its mechanistic role in neurodegeneration.

Findings: We identified the ovary-orientated protein OCIAD1 (Ovarian-Carcinoma-Immunoreactive-Antigen-Domain-Containing-1) as a neurodegeneration-associated factor for AD. Higher levels of OCIAD1, found in vulnerable brain areas and dystrophic neurites, were correlated with disease severity. Multiple early AD pathological events, particularly Aβ/GSK-3β signaling, elevate OCIAD1, which in turn interacts with BCL-2 to impair mitochondrial function and facilitates mitochondria-associated neuronal injury. Notably, elevated OCIAD1 by Aβ increases cell susceptibility to other AD pathological challenges.

Interpretation: Our findings suggest that OCIAD1 contributes to neurodegeneration in AD by impairing mitochondria function, and subsequently leading to neuronal vulnerability, and synaptic damages.

Funding: Ting Tsung & Wei Fong Chao Foundation, John S Dunn Research Foundation, Cure Alzheimer's Fund, and NIH R01AG057635 to STCW.

Keywords: Alzheimer's disease; Hyperamyloidosis; Mitochondrial dysfunction; Neurodegeneration; OCIAD1.

Conflict of interest statement

Declaration of Competing Interest The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Comparative studies identify OCIAD1 as a common factor associated with neurodegeneration in both Tg AD mice and AD patients. (a) The experimental workflow implementing integrative biology to identify and evaluate the potential factor and validate its role in AD. (b) Synaptosomes isolated from mouse cortical cortex were characterized via electronic microscopy (EM) (upper left, Scale bar = 200 nm) and examined via 2D-DIGE and MS-MALDI/TOF (upper middle and right). Proteomics results overlapped with the differential gene expression between vulnerable brain regions (EC and HIP) and invulnerable region (VCX) from sporadic AD patients, as shown by the heatmap. OD1, OCIAD1; Red (upregulation), blue (down-regulation). (c-d) OD1 protein levels are changed in the synaptosome from AD mice (APP/PS1-ΔE9, c; **P < 0.01 vs. control, student t-test), and in the forebrain from 5XFAD mice (d), *P < 0.05, **P < 0.01 vs. control (n = 3, student t-test), WT, wild type; Tg, transgenic mice. 2 M, mice at age of 2 months; 4 M, mice at age of 4 months. (e) OD1-positive cells were visualized by IHC staining in the frontal lobe of normal subject (NC) and AD. The loss of OD1-positive cells was compared to the reduction in cortex thickness in the frontal lobe of AD (n = 11–30, Scale bar = 15 µm). (f) Increased OD1 intensity is correlated with a decreased soma size in OD1-(+) cells in the FTL of AD patient (n = 180 in Non-AD and 308 in AD group). Data are presented as mean±SEM, *P < 0.05, **P < 0.01 vs. Non-AD (student t-test). (See also Fig. S1).
Fig. 2
Fig. 2
Brain OCIAD1 level is associated with AD development and under regulation of Aβ/GSK3β/p53 signals. (a) Workflow for analysis of OD1(OCIAD1) gene expression in human brain. (b-c) Higher expression level of OD1 was shown in the vulnerable brain sites in normal human subjects (left, in b) and AD patients (right, in b) while the increased level of OD1 correlates with severity of AD (c). (d-e) OD1 regulators were derived from GEO database to build PPI-based connection network (e) and generate AD-relevant regulatory network (f). (See also Table 1). (g-h) Elevated APP (g) or knockdown GSK-3β (h) change OD1 level in cells. APP-staining (red), OD1-staining (green), DAPI-staining (blue). APP mutant (APP-Mt), APP wild type (APP-Wt), wild type (NC), transgenic mice (FAD), knockdown GSK3β via sRNAi (GSK3β RNAi). (See also Fig. S2a-2c). (i) Chemical intervention of Aβ-GSK-3β signal affected levels of β-catenin and OD1 in cultured primary neurons. (j) Modulating β-catenin changed the OD1 level in HEK293 cells as shown in GEO data. (k) Knockdown (KO), over-expression (OE), knockdown β-catenin via shRNAi (sh-β-catenin). (l) Knockdown p53 reduces OD1 level in HEK293. Knockdown p53 via sRNAi (sRNAi). (See also Fig. S2d-2e). Data are presented as mean±SEM in (b–c, g–l). *P < 0.05, **P < 0.01 vs. control in (b) (student t-test, left panel, n = 32; right panel, n = 5), in c (one-way ANOVA, left panel, n = 3; Pearson Correlation Coefficient, right panel, n = 30), in h-l (student t-test, n = 3 in h-j, l, n = 6 in k),.*P < 0.05 vs. wild type in g (student t-test, n = 33 in APPWT, n = 45 in APPMt, and n = 3 in NC and FAD). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Integrative neurobiology study reveals a role of OCIAD1 in mitochondria-associated cell death. (a) Workflow for predicting OD1 (OCIAD1) function. (b) Cell death pathway and mitochondria are predicted as functional targets for OD1. Predicted functional partners of OD1 are indicated as nodes relevant to cell death pathways (blue), to mitochondria (orange), or both (green). (c-d) OD1 in the mitochondria was visualized via dual immunofluorescence staining against mitochondrial protein marks (SOD2) or Ds-Red Mito (c), and compared between the cerebral cortex of transgenic Tg AD mice (APP/PS1-ΔE9) and wild type mice (WT, d) at 4 months. Anti-OD1 (green), anti-SOD2 or DsRed (red). (e) Workflow for in vitro validation of OD1 function in mitochondria-associated cell death. (f-h) OD1 modulation affects cell injury in primary neurons induced by Aβ1-42 (f), in HT22 cells by MG132 (g), staurosporine (STR), H2O2 or glutamate (h). Cells infected with control vector encoding GFP only (CON, Mock, pCDH, pSIH), or plasmid encoding OD1 and GFP (OD1 or pCDH-OD1), shRNAi against OD1 (pSIH-OD1). (See also Fig. S3a-3d). (i) Up-regulation or down-regulation (j) of OD1 modulates the intrinsic apoptosis induced by MG-132 in HT22 cells. (k-l) Elevating OD1 in HT22 cells facilitates Str-induced apoptosis and release of cytochrome C (Cytc, k), SMAC (cyto-SMAC), EndoG (cyto-EndoG), but does not affect Caspase-8 activation (CASP8, l). Control vector (Mock), plasmid encoding OD1 (OD1). MG-12 h, MG-132 treatment for 12 hrs; STR treatment for 0 hr (0H), 4 hrs (Str4H), 8 hrs (Str8H), or 12 hrs (STR-12H). Data are presented as mean±SEM in (d-g) and (i- l)(student t-test, n = 3). * P < 0.05 vs. WT in (d) and vs.Veh in (f) #P < 0.05, ##P < 0.01 vs. Con in (g-h); *P < 0.05, **P < 0.01 vs. Con in (i-j); *P < 0.05, **P < 0.01 vs. Mock (k) or 0H (l), #P < 0.05 vs. Mock (l). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Upregulation of OCIAD1 impairs MMP via interaction with BCL-2. (a) Workflow for mechanistic study of OD1 (OCIAD1) in mitochondria-associated cell death. (b) OD1 elevation impairs MMP in HT22 cells before and after Str treatment. Cell transfected with empty vector (Mock), or plasmid encoding mouse OD1 (OD1). (See also Fig. S4a). (c-d) Effects of elevated OD1 on CCCP-induced cell injury, cleaved PARP (c), and CytC release (d) in HT22 cells. (e) PPI-based network analysis indicates BCL-2 as a key node linking OD1 to mitochondrial cell death pathway. (f) OD1 elevation interrupts the mitochondrial BCL-2/BAX ratio. Empty vector (Mock) or plasmid encoding OD1 (OD1), cytosolic fraction (Cyto), or mitochondrial fraction (Mito). (See also Fig. S4b-4d). (g) GST-pulldown assays verify BCL-2 as an interactor of OD1. (h) Dual luciferase assays show the interaction between BCL-2 and OD1, which disturbs BCL-2/BAX but facilitates BAX/BAX interaction. (i)In vitro incubation of OD1 with different combinations of BCL-2 and BAX in the isolated mitochondria from mouse cerebral cortex aggravates CytC releasing in the presence of CCCP. Data are presented as mean ± SEM in (b, f, h-i) (n = 3). * P < 0.05, ** P < 0.01 vs Veh, # P < 0.05, ## P < 0.05 vs. Mock in (b, f); ** P < 0.01 vs. Mock in (c-d). *P < 0.05, ** P < 0.01 vs. BCL-2 or vs BAX/BAX; ## P < 0.01 vs. BAX/BCL-2 in (h). * P < 0.05 and ** P < 0.01 vs. BCL-2/BAX/BSA, ## P < 0.01 vs. BAX/BSA in (i).
Fig. 5
Fig. 5
OCIAD1 mediates the long-term effects of Aβ aggregates on neurodegeneration in Alzheimer's disease by facilitating cell injury and neurite dysfunction.(ac) Pretreatment with a sub-lethal level of Aβ (1 μM, 10 hrs, preAβ) aggravated the cell injury induced by sequential stressors (a, MG-132, STR or CCCP), and facilitated caspase-3 activation induced by Str (b). Knockdown OD1 (c, siOD1) alleviated cell injury and caspase-3 activation in HT22 cells (b). **P < 0.01 vs. normal control (siCON), #P < 0.05 and ##P < 0.01 vs. experimental control (siCON-preAβ). (d) CCCP-induced CYTC releasing and caspase-3 activation in isolated mitochondria or synaptosome from the cerebral cortex of Tg AD mice (APP-PS1-ΔE9) in the presence of OD1.*P < 0.05 and **P < 0.01 vs. control treated with bovine serum albumin (BSA). β-tubulin (β-tub). (e-m) OD1 signal (red), YFP-labeled neuron and neurite (yellow) and neurite density (SMI-32, magenta) were shown in the area adjacent to amyloid plaque (X04, blue) in brain sections of the 5xFAD:YFP mice (5 M). Less neurite density, higher OD1 signal and ratio of OD1-postive dystrophic neurite was indicated by arrows in the circle with dashed line. OD1: OCIAD1, DNT: dystrophic neurite. **P < 0.01 vs. control X04- (area with no amyloid plaque and neurite loss). (n) A schematic summary of the mechanistic role of OD1 in mitochondria-associated neurodegeneration in AD. Early stage pathological changes (ie. Aβ aggregates) elevate OD1 through multiple pathways, including GSK3β. Upregulated OD1 leads to mitochondrial dysfunction through binding BCL-2, disturbing BCL-2/BAX interaction and impairing MMP (ΔΨm). In the presence of late stage cell injury stress, susceptible mitochondria release apoptotic factors to initiate cell death or activate caspase-3 causing non-apoptotic synaptic changes.

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