Multiple system atrophy and apolipoprotein E

Abstract

Objective

This study evaluated genetic associations of APOE alleles with risk of MSA and α-synuclein pathology, and also examined whether apoE isoforms differentially affect α-synuclein uptake in oligodendrocytes cell.

Methods

168 pathologically-confirmed MSA patients, 89 clinically-diagnosed MSA patients, and 1277 control subjects were genotyped for APOE. Human oligodendrocyte cell lines were incubated with α-synuclein and recombinant human apoE, with internalized α-synuclein imaged by confocal microscopy and cells analyzed by flow cytometry.

Results

No significant association with risk of MSA or was observed for either APOE ε2 or ε4. α-synuclein burden was also not associated with APOE alleles in the pathologically-confirmed patients. Interestingly, in our cell assays, apoE ε4 significantly reduced α-synuclein uptake in the oligodendrocytic cell line.

Conclusions

Despite differential effects of apoE isoforms on α-synuclein uptake in a human oligodendrocytic cell, we did not observe a significant association at the APOE locus with risk of MSA or α-synuclein pathology.

Introduction

Widespread presence of glial cytoplasmic inclusions (GCIs) is the neuropathologic hallmark of MSA.¹ GCI is the accumulation of α-synuclein protein in the cytoplasm of oligodendrocytes, the myelin-producing support cells in CNS. Recently, dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes has been associated with the unique neuropathology of MSA.² Apolipoprotein E (APOE) is a well-established lipid-metabolism gene. The APOE ε4 allele is the major genetic determinant of late-onset Alzheimer’s disease (AD) risk and has also been strongly associated the synucleinopathy dementia with Lewy bodies³, whereas APOE ε2 is known as a protective factor for AD and dementia.³ Compared to other more common neurodegenerative diseases, little is known about the genetics of MSA; recent reports have nominated variants in several genes (SNCA, MAPT, LRRK2, COQ2, GBA) as potential risk factors, though validation is needed.⁴ We hypothesized that the APOE alleles could play a role in the pathogenesis of MSA. In this study, we first assessed associations of the APOE ε4 and ε2 alleles with risk of MSA and with α-synuclein burden. We also investigated whether apolipoprotein E (apoE) differentially affected α-synuclein uptake in a human oligodendrocytic cell line in an isoform-dependent manner.

Methods

Study subjects

168 pathologically-confirmed MSA patients⁵, 89 clinically-diagnosed MSA patients¹, and 1277 controls were included (Supplemental Table 1). The pathologically-confirmed patients were all available cases obtained from the Mayo Clinic brain bank for neurodegenerative disorders in Jacksonville, FL, and were diagnosed by a single neuropathologist (D.W.D.).⁵ Both clinically-diagnosed MSA patients and control subjects were seen at the Mayo Clinic in Jacksonville, FL (MSA: N=54, Controls: N=712) and Rochester, MN (MSA: N=35, Controls: N=565). All controls were neurologically normal and free of a family history of a movement disorder. All subjects were unrelated non-Hispanic Caucasians. The primary comparison was between the pathologically-confirmed MSA patients and controls. As a secondary comparison, we combined the pathologically-confirmed and clinically-diagnosed MSA patients for comparison with controls.

Pathological analysis

Immunohistochemistry for α-synuclein (NACP, Mayo Clinic antibody, Jacksonville, FL)⁶ was conducted to establish the neuropathological diagnosis.⁵ The burden of α-synuclein in striatopallidal fibers was measured quantitatively as described previously and was available for 130 of the pathologically-confirmed MSA patients.⁷ Braak neurofibrillary tangles stage (available for 163 patients) and Thal amyloid phase (available for 158 patients) were assigned to each case with thioflavin S fluorescent microscopy.^{8, 9}

Genetic Analysis

Genomic DNA was extracted from peripheral blood monocytes or frozen brain tissue using the standard protocols. Genotyping for APOE alleles (rs429358 C/T and rs7412 C/T) was performed using a custom TaqMan Allelic Discrimination Assay on an ABI 7900HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) (primer sequences are available upon request).

Statistical analysis

Associations of presence of the APOE ε4 and ε2 alleles with risk of MSA were evaluated using logistic regression models that were adjusted for age and gender. Additionally, we also compared the ε3/ε3 genotype to the ε3/ε4 genotype to directly assess the effect of ε4, and similarly we compared the ε3/ε3 genotype to the ε2/ε3 genotype to directly assess the effect of ε2. Finally, we utilized Fisher’s exact test to perform a general comparison of APOE genotype.

In pathologically-confirmed MSA patients, we assessed associations of ε4 and ε2 with α-synuclein burden in striatopallidal fibers, Braak stage, and Thal phase using linear regression and proportional odds logistic regression models that were adjusted for age at death and gender. Given that statistical tests of association were performed for both APOE ε4 and ε2, P≤0.025 was considered as statistically significant after Bonferroni correction. With 168 pathologically-confirmed MSA patients and 1269 controls included in our primary analysis, we had 80% power at the P≤0.025 significance level to detect odds ratios of 1.75 (association with ε4) and 1.90 (association with ε2) in relation to risk of MSA. All statistical analysis was performed using SAS.

Materials

Human oligodendrocytic cell line, MO3.13, was purchased from Cedarlane Labs (Burlington, NC). Recombinant human α-synuclein, HiLyte^™ Fluor 488 labeled was from Anaspec (Fremont, CA). Recombinant human apoE proteins were from Fitzgerald Industries (Acton, MA).

Flow cytometry analysis of α-Synuclein uptake

Human oligodendrocytic cell line, MO3.13, were cultured and treated with 10 nM of fluorescently labeled recombinant human α-synuclein with or without 50 nM of recombinant human apoE for 18 hours. Total of 10,000 cells were analyzed by FACS Accuri (BD Bioscience). The median fluorescence signals in each condition were quantified using CFLOW^® plus software (BD Bioscience), and analyzed by one-way ANOVA with Tukey’s post-hoc analysis.

Confocal analysis of α-Synuclein uptake by MO3.13

MO3.13 were plated on coverslips and treated with 250 nM α-synuclein HiLyte 488 and 1.25 μM of apoE for 18 hours. At the end of the treatment, LysoTracker® Red (Life Technologies) was added to label lysosomes. The images were acquired using confocal laser-scanning fluorescence microscope (LSM 510, Carl Zeiss).

Results

There was no significant association of APOE ε4 or ε2 with risk of MSA (Table 1) or risk of MSA subtypes (Supplemental Tables 2 and 3). Similarly, when making a general comparison of APOE genotype with controls, no difference was observed for the pathologically-confirmed MSA patients (P=0.78) or the combined MSA series (P=0.47) (Supplemental Table 4). There was no association between α-synuclein burden in the striatopallidal fibers and presence of either APOE ε4 (P=0.71) or ε2 (P=0.49). As shown in Supplemental Table 5, in the pathologically-confirmed MSA series, ε4 was associated with a significantly higher Thal phase (P<0.0001) but was not associated with Braak stage (P=0.25), while ε2 was not associated with either outcome after multiple testing adjustment (P≥0.047) though non-significant trends toward less AD pathology in ε2 carriers were noted.

TABLE 1.

Associations of APOE ε4 and ε2 with risk of MSA

	Association between APOE ε4 and risk of MSA
Comparison/Disease group	N	No. (%) with APOE ε4	OR (95% CI)	P-value
Presence vs. absence of APOE ε4
Controls	1269	318 (25.1%)	1.00 (reference)	N/A
Pathologically-confirmed MSA patients	168	37 (22.0%)	0.79 (0.53, 1.17)	0.24
All MSA patients (pathologically-confirmed and clinically-diagnosed)	257	57 (22.2%)	0.78 (0.56, 1.08)	0.13
Comparison of ε3/ε3 and ε3/ε41
Controls	1044	260 (24.9%)	1.00 (reference)	N/A
Pathologically-confirmed MSA patients	138	33 (23.9%)	0.90 (0.59, 1.36)	0.61
All MSA patients (pathologically-confirmed and clinically-diagnosed)	214	51 (23.8%)	0.86 (0.61, 1.23)	0.42
	Association between APOE ε2 and risk of MSA
Comparison/Disease group	N	No. (%) with APOE ε2	OR (95% CI)	P-value
Presence vs. absence of APOE ε2
Controls	1269	200 (15.8%)	1.00 (reference)	N/A
Pathologically-confirmed MSA patients	168	29 (17.3%)	1.13 (0.73, 1.73)	0.59
All MSA patients (pathologically-confirmed and clinically-diagnosed)	257	42 (16.3%)	1.07 (0.74, 1.54)	0.73
Comparison of ε 3/ε3 and ε2/ε32
Controls	948	164 (17.3%)	1.00 (reference)	N/A
Pathologically confirmed MSA patients	131	26 (19.8%)	1.18 (0.74, 1.87)	0.50
All MSA patients (pathologically-confirmed and clinically-diagnosed)	199	36 (18.1%)	1.06 (0.71, 1.59)	0.77

OR=odds ratio; CI=confidence interval. ORs, 95% CIs, and p-values result from logistic regression models adjusted for age (age at death for pathologically-confirmed MSA patients, age at MSA onset for clinically-diagnosed MSA patients, and age at blood draw for controls) and gender.

¹For comparison of the ε3/ε3 and ε3/ε4 genotypes, ORs correspond to the ε3/ε4 genotype, and individuals with APOE genotypes other than ε3/ε3 or ε3/ε4 (i.e. ε2/ε2, ε2/ε3, ε2/ε4, and ε4/ε4) were excluded.

²For comparison of the ε3/ε3 and ε2/ε3 genotypes, ORs correspond to the ε2/ε3 genotype, and individuals with APOE genotypes other than ε3/ε3 or ε2/ε3 (i.e. ε2/ε2, ε2/ε4, ε3/ε4, and ε4/ε4) were excluded.

In our cellular assay, the human oligodendrocytic cell line, MO3.13, showed reduced colocalization of fluorescently-labeled α-synuclein and lysoTracker when co-treated with recombinant apoE ε4 compared to apoE ε2, ε3, or bovine serum albumin (Fig. 1A). The flow cytometry analysis further confirmed the significantly reduced internalization of fluorescently-labeled α-synuclein by MO3.13 when co-treated with apoE ε4 (Fig. 1B and 1C).

Figure 1. ApoE4 reduces α-Synuclein uptake by oligodendrocytic cell line.

(A) Confocal analysis of α-Synuclein (green) internalization by MO3.13 co-treated with apoE or BSA. Blue: DAPI, red: LysoTracker®. Scale bar= 20 μm. (B) Flow cytometry analysis of MO3.13 incubated with α-Synuclein and apoE (E2: green, E3: blue, E4: red) or BSA (purple). (C) The median fluorescence signals in each condition. Data plotted as mean ± SD (N= 4, One-Way ANOVA with Tukey’s post-hoc analysis, * p< 0.05, ** p< 0.01).

Discussion

The results of our genetic study showed that neither the APOE ε4 nor ε2 alleles were notably associated with risk of MSA. These findings are in line with the results of previous small studies (n= 22; 59; 47; 40 and 12)^10–14 and no signal at the APOE locus in the recent genome-wide association study of 331 pathologically-confirmed MSA patients.¹⁵ It also is worth noting that in agreement with these findings, a recent study reported no association between rapid eye movement sleep behavior disorder (which could be a clinical prodromal feature preceding the development of MSA) and risk of MSA.¹⁶

Interestingly, in our oligodendrocytic cellular assays, we showed that the apoE ε4 isoform significantly reduced α-synuclein uptake, which means apoE regulates α-synuclein uptake in an isoform-dependent manner. The mechanisms underlying this observation remains to be investigated. Notably, oligodendrocytes have little endogenous α-synuclein,¹⁷ nevertheless GCIs (the pathological hallmark of MSA) consist of α-synuclein. One possible mechanism is that oligodendrocytes might take up α-synuclein from the extracellular environment to make GCIs in the brain of MSA.¹⁷ Based on our results, we could hypothesize that apoE ε4 may be a disease progression modifier for MSA by reducing the uptake of α-synuclein by oligodendrocytes. Although the lack of association between ε4 and risk of MSA in our genetic association analysis does not initially seem to support this result, it is important to highlight that although not statistically significant, the direction of the association that we observed was protective (OR=0.78), and based on 95% confidence limits this could plausibly be as low as 0.56; the possibility of a false-negative association is important to consider. Given our functional data, further studies may be warranted to investigate whether APOE allelic variation plays a role in MSA susceptibility or progression.