Effect of ApoE isoforms on mitochondria in Alzheimer disease

Abstract

Objective

To test the hypothesis that ApoE isoforms affect mitochondrial structure and function that are related to cognitive impairment in Alzheimer disease (AD), we systematically investigated the effects of ApoE isoforms on mitochondrial biogenesis and dynamics, oxidative stress, synapses, and cognitive performance in AD.

Methods

We obtained postmortem human brain tissues and measured proteins that are responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and sirtuin 3 [SIRT3]), for mitochondrial dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin-like protein 1 [DLP1]), for oxidative stress (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]), and for synapses (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]). A total of 46 cases were enrolled, including ApoE-ɛ4 carriers (n = 21) and noncarriers (n = 25).

Results

Levels of these proteins were compared between ApoE-ɛ4 carriers and noncarriers. ApoE-ɛ4 was associated with impaired mitochondrial structure and function, oxidative stress, and synaptic integrity in the human brain. Correlation analysis revealed that mitochondrial proteins and the synaptic protein were strongly associated with cognitive performance.

Conclusion

ApoE isoforms influence mitochondrial structure and function, which likely leads to alteration in oxidative stress, synapses, and cognitive function. These mitochondria-related proteins may be a harbinger of cognitive decline in ApoE-ɛ4 carriers and provide novel therapeutic targets for prevention and treatment of AD.

There are 3 isoforms of ApoE: ApoE-ɛ2, ApoE-ɛ3, and ApoE-ɛ4. ApoE has important roles in the pathophysiology of Alzheimer disease (AD). ApoE-ɛ4 is a major genetic risk factor for late-onset AD and is thought to be a driver of impaired cerebral physiology in AD.^1–3 Approximately 25% of the US population and 60% of patients with AD are ApoE-ɛ4 carriers. Although we do not fully understand how ApoE-ɛ4 causes neurodegeneration (with evidence of both amyloid-dependent and -independent mechanisms), accumulated evidence has strongly suggested that mitochondrial dysfunction plays a key role in AD pathogenesis.⁴ Mitochondrial dysfunction could lead to enhanced oxidative stress and has been identified as an early event in AD progression.⁵ Our previous research revealed that the regulation of mitochondrial protein such as sirtuin 3 (SIRT3) could affect AD pathogenesis.^6–8 ApoE-ɛ4 interacts with the mitochondrial genome in determining the outcome of acute brain injury.⁹ However, how ApoE isoforms affect mitochondrial proteins and their function within the context of AD pathogenesis remains unclear.

We hypothesized that mitochondrial proteins are influenced by ApoE isoforms and subsequently their altered function affects the pathophysiology of AD. In this study, we examined the expression of proteins responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and SIRT3) and dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin like protein 1 [DLP1]); oxidative stress proteins (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]); and the synaptic proteins (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]) in human postmortem brains of ApoE-ɛ4 carriers and noncarriers.

Methods

Patients

All patients were recruited from the Arizona Alzheimer's Disease Center Clinical Core.¹⁰ Clinical and neuropsychological assessment is performed annually according to the National Alzheimer's Coordinating Center protocol. Postmortem human brain tissues obtained from ApoE-ε4 carriers (n = 21) and noncarriers (n = 25) were autopsied at the Banner Sun Health Research Institute Brain and Body Donation Program.¹¹ The median postmortem interval is 3.2 hours for the entire collection. Frozen brain tissues of the middle temporal gyrus (MTG) were homogenized and centrifuged, and supernatants were collected for further analysis.

Patients designated as “cognitively normal” (CN) have no cognitively based limitations in daily living activities and a Clinical Dementia Rating (CDR) score of 0. The diagnosis of mild cognitive impairment (MCI) was given according to published consensus criteria¹² and a CDR score of 0.5. A diagnosis of probable AD by the National Institute of Neurological Disorders and Stroke criteria¹³ was given to patients with a CDR score ≥1.0 and who had severe impairment of learning and delayed recall. In addition, they were free of other neurodegenerative disorders. AD pathology was confirmed by cortical Aβ neuritic plaque density (plaque score) and Braak tangle stage by a neuropathologist (T.G.B.). Because protein levels may change with age, only the final antemortem assessment score within 1 year before death, if available, was included for the correlation analysis. The raw scores from the Mattis Dementia Rating Scale (DRS), Auditory Verbal Learning Test total learning (AVLT-TL), and 60-item Boston Naming Test (BNT) were converted to age- and education-corrected z scores.

Standard protocol approvals, registrations, and patient consents

We received approval from an ethical standards committee on human experimentation (the Western Institutional Review Board) for studies using human patients. We also received written informed consent obtained from all patients (or guardians of patients) participating in the study (consent for research).

Protein ELISA

Frozen brain tissues were homogenized and sonicated in radioimmunoprecipitation assay buffer plus protease inhibitors on ice. The supernatant was collected for ELISA. Rabbit unlabeled capture antibody (100 µL) (Cell Signaling, Danvers, MA) in coating buffer was added into 96 wells of a high-affinity protein-binding ELISA plate (BioLegend, San Diego, CA) and incubated in a refrigerated room (4°C) overnight. After the plate was washed 4 times using phosphate-buffered saline/0.05% Tween 20, 1% casein (Life Technologies, Carlsbad, CA) was used as the blocking buffer for 1 hour at room temperature. Human SIRT3 protein (Sigma, St. Louis, MO) was used as the standard control with the following dilution series: 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, and 0 ng/mL. Samples and standards were incubated at 4°C overnight. Unlabeled goat antibody (Sigma) was used as the detection antibody and incubated at room temperature for 2 hours. Anti-goat secondary antibody–conjugated horseradish peroxidase (Life Technologies) and tetramethylbenzidine (Sigma) were used to magnify the signal. Color development was finally stopped by adding 2N sulfuric acid, and the plate was read immediately at 450 nm on Tecan Infinite M200 Pro Spectrometry. The SIRT3 protein level (pg/µg) was normalized with total protein.

Western blot

The brain tissues (temporal cortex) were homogenized in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Rockford, IL) with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) on ice. Total protein concentrations were determined using the BCA protein assay kits (Thermo Fisher Scientific). Western blot was performed with 50 µg total protein, and proteins were separated on 12% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories, Hercules, CA) that were subsequently transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The following primary antibodies were used: anti-SIRT3 (#5490S; Cell Signaling Technology Inc., Danvers, MA), anti–PGC-1α (#NBP1-04676; Novus Biologicals Inc., Littleton, CO), anti-SOD2, anti-Foxo3a, anti-MFN1, anti-MFN2, anti-DLP1, anti-PSD95, anti-Syn1, anti–β-actin (Santa Cruz, Dallas, TX), IRDye 800CW, and IRDye 680CW antibodies (LI-COR, Lincoln, NE). Immunoreactive signals were quantified using Odyssey CLx. Protein levels were presented with percentage relative to β-actin, an internal control.

Statistics

One-way analysis of variance with the post hoc Tukey test was applied in GraphPad Prism version 5.03 for the data from multiple groups. Pearson correlation analysis was used for the associations between all parameters and neuropsychological test scores. All statistics were performed using IBM SPSS Statistics version 22. Statistical significance was defined as p < 0.05 for all analyses.

Data availability

Data are available on reasonable request.

Results

Demographics of patients

Patients' demographic information was summarized in the table. Twenty-one ApoE-ɛ4 carriers and 25 noncarriers were enrolled in the study. Except Mini-Mental State Examination (MMSE), the average age, education, and sex were similar between ApoE-ɛ4 carriers and noncarriers. MMSE scores were lower in ApoE-ɛ4 carriers. In addition, both groups had similar numbers of CN, patients with MCI, and patients with AD.

Table.

Demographic data

Mitochondrial biogenesis and dynamics were influenced by ApoE isoforms

PGC-1α is considered the master regulator of mitochondrial biogenesis. Its activation increases mitochondrial mass and overall function. SIRT3 is its downstream substrate. We compared SIRT3 and PGC-1α levels in ε4 carriers vs noncarriers. ELISA data showed that SIRT3 was significantly lower in the temporal cortices in ε4 carriers compared with noncarriers (ε4 carriers 43.6 ± 2.4 pg/µg vs noncarriers 55.8 ± 3.1 pg/µg, p = 0.004, figure 1A), a 30.8% reduction. PGC-1α protein levels showed a similar degree reduction in ε4 carriers compared with noncarriers (67.2 ± 5.5% vs 87.9 ± 3.9%, p = 0.003, figure 1B). Further analysis indicated that there was a significant correlation between PGC-1α and SIRT3 (r = 0.459, p < 0.01, figure 1C).

Figure 1. Mitochondrial biogenesis and dynamics were influenced by ApoE isoforms.

Human postmortem brain tissues (middle temporal gyrus) were collected to compare ApoE-ε4 noncarriers (n = 25, dot) vs carriers (n = 21, square). (A) ELISA data of SIRT3 protein levels were normalized with total protein (pg/µg); (B) PGC-1α levels were normalized with β-actin in Western blot. (C) PGC-1α showed a correlation with SIRT3. (D) Representative Western blot bands for mitofusin 1 (MFN1), mitofusin 2 (MFN2), and dynamin-like protein 1 (DLP1) were shown. (E) MFN1, (F) MFN2, and (G) DLP1 protein levels were plotted to compare carriers vs noncarriers. β-actin was used as an internal control. **p < 0.01, carriers vs noncarriers. Patients were divided into 3 groups: cognitively normal controls (CN, n = 14), mild cognitive impairment (MCI, n = 16), and AD (n = 16). Data of scatter plots were compared for (H) MFN1, (I) MFN2, and (J) DLP1. #p < 0.05, CN vs AD; ∆∆p < 0.01, MCI vs AD. AD = Alzheimer disease.

Mitochondrial fusion and fission play a central role in the pathologic process of several neurodegenerative disorders.¹⁴ Three proteins involved in fusion and fission were measured in the human brain. Levels of these 3 proteins were lower in human ApoE-ɛ4 carriers than noncarriers (carriers vs noncarriers, MFN1: 12.1 ± 0.7 vs 16.1 ± 1.1, p < 0.01; MFN2: 49.5 ± 3.9 vs 66.3 ± 3.8, p < 0.01; DLP1: 167.6 ± 12.6 vs 215.5 ± 12.3, p < 0.01) (figure 1, D–G).

Previous studies indicated that mitochondrial dynamics could be affected through impaired mitochondrial fission and fusion induced by β-amyloid or amyloid precursor protein in AD models.^15,16 Thus, we compared mitochondrial dynamic proteins across cases with different levels of cognitive difficulty including AD, MCI, and CN. Levels of all 3 proteins (MFN1, MFN2, and DLP1) were all reduced in patients with AD compared with CN and patients with MCI (CN, MCI, and AD, MFN1: 15.1 ± 1.1, 16.9 ± 1.5, and 10.8 ± 0.6, CN vs AD, p < 0.05, MCI vs AD, p < 0.01; MFN2: 65.3 ± 4.1, 65.4 ± 5.7, and 46.1 ± 3.7, CN vs AD, p < 0.05, MCI vs AD, p < 0.05; DLP1: 203.7 ± 14.8, 227.5 ± 15.8, and 151.0 ± 12.0, CN vs AD, p < 0.05, MCI vs AD, p < 0.01) (figure 1, H–J).

Mitochondrial proteins were strongly correlated with cognitive function

Mitochondria play critical physiologic functions in neurons.¹⁷ Impaired mitochondrial biogenesis and function is involved in many neurodegenerative diseases and damages synaptic neurotransmission, which is critical for normal cognitive function.¹⁸ We further analyzed whether there were correlations between these proteins and cognitive performance. SIRT3, PGC-1α, MFN1, and DLP1 were strongly correlated with DRS Z scores, a global assessment of cognitive function (figure 2, A–D); SIRT3, PGC-1α, and MFN1 were significantly associated with the BNT (60 items) Z score, an assessment of frontal and temporal brain functions (figure 2, E–H), and the AVLT-TL Z score, an assessment of temporal lobe function (figure 2, I–L).

Figure 2. Mitochondrial proteins were correlated with cognitive function.

Data of mitochondrial protein levels were obtained from ApoE-ɛ4 carriers (n = 21, square) and noncarriers (n = 25, dot). The Pearson correlation curve was depicted. The DRS Z score was correlated with (A) SIRT3, (B) PGC-1α, (C) mitofusin 1 (MFN1), and (D) DLP1. The Boston Naming total (60 items) Z score was correlated with (E) SIRT3, (F) PGC-1α, (G) MFN1, and (H) DLP1. The AVLT total learning Z score was correlated with (I) SIRT3, (J) PGC-1α, (K) MFN1, and (L) DLP1. AVLT = Auditory Verbal Learning Test; DRS = Dementia Rating Scale.

Mitochondrial oxidative stress was influenced by ApoE isoforms

Mitochondria regulate cell functions through not only energy generation but also reactive oxygen species production. The oxidative stress proteins SOD2 and Foxo3a were measured. In human brain samples, levels of SOD2 and Foxo3a were lower in ApoE-ɛ4 carriers than noncarriers (carriers vs noncarriers: SOD2, 54.4 ± 2.7 vs 67.5 ± 3.7, p < 0.01; Foxo3a, 19.2 ± 1.1 vs 30.5 ± 1.4, p < 0.01) (figure 3, A–C).

Figure 3. Mitochondrial oxidative stress was influenced by ApoE isoforms.

Human brain tissues (middle temporal gyrus) were collected from ApoE-ɛ4 noncarriers (n = 25, dot) and carriers (n = 21, square). (A) Representative Western blot bands for superoxide dismutase 2 (SOD2) and forkhead-box protein O3a (Foxo3a) were shown. (B) SOD2 and (C) Foxo3a levels were plotted to compare carriers vs noncarriers. β-actin was used as an internal control. **p < 0.01, carriers vs noncarriers.

Synaptic plasticity was influenced by ApoE isoforms

Increasing evidence suggests that mitochondrial abnormalities are involved in the loss of synapses, defective axonal transport, and cognitive decline in patients with AD.¹⁹ We investigated how ApoE isoforms affected the expression of synaptic proteins in the temporal lobe. Levels of PSD95 and Syn1 were both downregulated in ApoE-ɛ4 carriers compared with noncarriers (carriers vs noncarriers: PSD95, 150.2 ± 12.9 vs 195.6 ± 10.2, p < 0.01; Syn1, 212.0 ± 14.2 vs 286.9 ± 13.4, p < 0.01 (figure 4, A–C). Not surprisingly, Syn1 showed a significant correlation with cognitive performance on DRS, BNT, and AVLT (figure 4, D–F).

Figure 4. Synaptic plasticity was influenced by ApoE isoforms.

Human brain tissues (middle temporal gyrus) were collected from ApoE-ɛ4 noncarriers (n = 25, dot) and carriers (n = 21, square). (A) Representative Western blot bands for postsynaptic density protein 95 (PSD95) and synapsin 1 (Syn1) were shown. (B) PSD95 and (C) Syn1 levels were plotted to compare carriers vs noncarriers. β-actin was used as an internal control. **p < 0.01, carriers vs noncarriers. The Pearson correlation curve was depicted. Syn1 was correlated with the (D) DRS Z score, (E) Boston Naming total (60 items) Z score, and (F) AVLT total learning Z score. AVLT = Auditory Verbal Learning Test; DRS = Dementia Rating Scale.

Discussion

In this study, we demonstrated that ApoE-ɛ4 downregulated mitochondrial biogenesis, dynamics, and its antioxidative stress proteins. ApoE-ɛ4 was associated with reduced synaptic proteins and impaired cognitive function. Since a mitochondrial cascade hypothesis was proposed in 2004,²⁰ it is increasingly suspected that mitochondrial dysfunction plays a detrimental role in AD.²¹ Mitochondrial dysfunction has been identified as an early event in AD pathogenesis,⁵ and enhancing mitochondrial function may attenuate pathologic changes in AD.^4,6–8

Mitochondrial biogenesis is activated by environmental stimuli or cellular stress, so it serves as a protective mechanism for the cell.^22,23 PGC-1α, a member of the PGC family, is the key player in mitochondrial biogenesis.^23,24 It is known to activate substrate proteins that are responsible for transcribing nuclear-encoded mitochondrial proteins and post-translational protein modification (SIRT3).^24,25 Although PGC-1α may not be required for normal development of mitochondria, lack of PGC-1α exhibits decreased ability to adapt to physiologic stress.^26,27 PGC-1α is a transcriptional activator that regulates SIRT3 expression at both mRNA and protein levels.^28,29 Exercise and fasting can activate PGC-1α–SIRT3 signal pathway and subsequently promote increased mitochondrial function.^30–32 We demonstrate a strong correlation between PGC-1α and SIRT3 expression. Several factors can lead to hypometabolism, including sirtuin pathway abnormalities. Previous studies reported that SIRT3 noticeably declines in the hippocampal and frontal lobe regions of AD brains in humans and mice.^6,8,33,34 This study demonstrated that ApoE-ɛ4 impaired mitochondrial biogenesis by reducing these 2 key players.

The shapes of mitochondria change continuously through a combination of fission, fusion, and motility. Mitochondrial dynamics involves fission and fusion of mitochondrial outer and inner membranes. Strong evidence has emerged to implicate disturbed mitochondrial fission and fusion as a central pathologic process in several neurodegenerative disorders.¹⁴ MFN1, MFN2, and DLP1 are important proteins regulating mitochondrial fusion and fission, respectively.^35,36 Fusion assists in modifying metabolic or environmental stress by enabling genetic complementation of slightly damaged mitochondria. Thus, these mitochondrial genomes generate all the necessary components for a functional mitochondrion. Without proper fusion, mitochondria would become fragmented as an individual ages. ApoE-ɛ4 decreases both fusion and fission proteins directly or indirectly that leads to mitochondrial damage, especially in the old age.

Mitochondria regulate cell functions through reactive oxygen species production. Damage to mitochondria could cause a decline in ATP production and overexposure to oxidative stress that would accelerate cell death.³⁷ The overexpression of mitochondria-specific protein increased ATP production and reduced oxidative stress.^8,38 ApoE-ɛ4 carriers have oxidative stress long before the symptomatic onset of AD and even before the diagnosis of MCI.³⁹ We have shown that oxidative stress was affected in an ApoE isoform–dependent manner.⁴⁰ This is consistent with our previous findings that impaired learning and memory abilities started in 12-month-old ApoE transgenic mice that later developed significant hippocampus and cortex atrophy at 18 months.^7,41

Synaptic integrity is closely related to cognitive function. Mitochondrial dysfunction is also found in early impairment of synaptic plasticity and cognitive decline in AD.¹⁹ Improving mitochondrial function could protect synaptic plasticity and cognitive impairment.^18,42 Our data suggested that ApoE-ɛ4 may affect synaptic plasticity through regulating mitochondrial function proteins. Furthermore, our data showed a close relationship between mitochondrial proteins, the synaptic protein, and cognitive function in AD.

There are limitations to this study. First, we used a single-center cohort. The patients may not reflect the full range of socioeconomic characteristics, which could affect cognitive function. But the genetic status and biological findings are unlikely to be affected by this. Our results should be further validated in other cohorts and interpreted in the appropriate population. Second, this study was based on cross-sectional data. There are age-related effects shown in animal models. Evidence has emerged showing that ApoE-ɛ4 affects morphologic changes in the brain as early as infant stage. Specifically, ApoE-ɛ4 carriers have lower gray matter volume and myelin water fraction in brain areas that are susceptible to AD-related pathology decades later.⁴³ In a population setting, we would likely see age-related changes in mitochondrial biogenesis and dynamics. Third, it would be interesting to see longitudinal changes, which would help to validate the finding and to address the question of conversion from preclinical dementia to dementia. Fourth, we examined only a small part of the brain. The target proteins were measured only in the MTG from the postmortem human brain in this study. In our previous studies, we tested our target proteins in multiple brain areas, including the MTG, superior frontal gyrus, primary visual cortex, and entorhinal cortex. Our target proteins were affected mostly in the MTG.^6,10,33,44 In future validation studies, we will include more areas of the brain.

In summary, ApoE isoforms influence mitochondrial biogenesis and dynamics, its antioxidative stress proteins, and synaptic proteins. These mitochondrial structure and function were strongly associated with cognitive function.

Glossary

AVLT-TL

Auditory Verbal Learning Test total learning

AD

Alzheimer disease

BNT

Boston Naming Test

CN

cognitively normal

CDR

Clinical Dementia Rating

DRS

Dementia Rating Scale

MTG

middle temporal gyrus

MCI

mild cognitive impairment

MMSE

Mini-Mental State Examination

SIRT3

sirtuin 3

Appendix. Authors

Study funding

This work is funded by the National Institute on Aging (P30 AG19610 Arizona Alzheimer's Disease Core Center), the Flinn Foundation (2190), and the Barrow Neurological Foundation (3032226). In addition, the Brain and Body Donation Program has been supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders).

Disclosure

J. Yin is funded by the Flinn Foundation (2190) and the Barrow Neurological Foundation (3032226). E.M. Reiman is funded by NIH grant P30AG19610 (Arizona Alzheimer's Disease Core Center), R01AG055444, and R01AG031581. T.G. Beach and G.E. Serrano are funded by NIH grant P30AG19610 (Core D: Neuropathology Core) and the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders). M.N. Sabbagh is funded by R01AG059008. M. Nielsen is funded by the Barrow Neurological Foundation (3032226). R.J. Caselli is funded by NIH grant P30AG19610 (Core B: Clinical Core) and R01AG031581. J. Shi is funded by the Flinn Foundation (2190) and the Barrow Neurological Foundation (3032226). Go to Neurology.org/N for full disclosures.