Baseline demographic and biomarker characteristics of the study participants
The demographic features and biomarker characteristics of cognitively normal type 2 diabetic patients, MCI patients, and AD dementia patients are shown in Table 1. The T2DM participants in the study included 41 cognitively normal controls, 97 individuals with MCI (68 individuals with sMCI; 29 individuals with pMCI) and 36 patients with AD dementia. There were no significant differences in age, sex, educational level, HbA1c or vascular risk factors among the groups. Compared with MCI (pMCI and sMCI) and cognitively normal participants, patients with AD dementia had lower MMSE and MoCA scores. For participants with baseline MRI measurements, total hippocampal volume and AD signature cortical thickness were lower in DM patients with pMCI (5.99 ± 0.79 cm3, 2.47 ± 0.13 mm) than in sMCI patients (6.39 ± 0.71 cm3, 2.55 ± 0.19 mm). Total hippocampal volume, AD signature cortical thickness, and total gray matter volume in AD patients (5.11 ± 0.84 cm3, 2.17 ± 0.33 mm, 548.9 ± 24.8 cm3, respectively) were lower than those in MCI patients (pMCI: 5.99 ± 0.79 cm3, 2.47 ± 0.13 mm, 607.5 ± 23.3 cm3, respectively, and sMCI: 6.39 ± 0.71 cm3, 2.55 ± 0.19 mm, 609.8 ± 22.4 cm3, respectively) and cognitively normal participants (7.24 ± 0.73 cm3, 2.59 ± 0.18 mm, 612.3 ± 27.5 cm3, respectively). The PVH in MCI (pMCI: 9028 ± 6726 mm3 and sMCI: 8376 ± 6026 mm3) and AD patients (11781 ± 8921 mm3) was higher than that in cognitively normal participants (6119 ± 4464 mm3). The exosomal concentrations of Aβ42, T-tau, and P-T181-tau in AD patients (4.15 ± 0.60, 207.2 ± 36.5, and 89.4 ± 22.0 pg/ml, respectively) were higher than those in MCI patients (pMCI: 3.78 ± 0.75, 184.7 ± 25.3, 66.8 ± 16.5 pg/ml, respectively, and sMCI: 3.38 ± 0.84, 167.7 ± 30.0, 57.3 ± 13.2 pg/ml, respectively) and cognitively normal participants (3.17 ± 0.77, 163.5 ± 34.1, 55.9 ± 10.2 pg/ml, respectively). Furthermore, the exosomal concentrations of Aβ42, T-tau, and P-T181-tau in pMCI patients were higher than those in sMCI patients and cognitively normal participants. Compared with MCI (pMCI: 530.6 ± 131.5 pg/ml and sMCI: 593.0 ± 130.4 pg/ml) and cognitively normal participants (627.6 ± 152.0 pg/ml), patients with AD dementia (461.4 ± 116.9 pg/ml) had lower exosomal concentrations of SNAP-25. The exosomal concentrations of SNAP-25 in pMCI patients were lower than those in sMCI and cognitively normal participants.
Table 1
Demographic and clinical characteristics of the participants in baseline
| CN (n = 41) | sMCI (n = 68) | pMCI (n = 29) | AD (n = 36) |
Age, years | 69.8 ± 7.1 | 71.0 ± 8.0 | 72.6 ± 7.7 | 72.0 ± 7.1 |
Education, years | 9.1 ± 4.9 | 8.1 ± 5.3 | 8.8 ± 4.5 | 8.0 ± 4.9 |
Gender, female (%) | 22 (53.6) | 41 (60.3) | 16 (55.2) | 20 (55.6) |
Duration of type 2 diabetes, years | 9.2 ± 3.6 | 9.6 ± 3.3 | 10.8 ± 4.2 | 11.0 ± 4.1a |
HbA1c (%) | 7.9 ± 1.4 | 7.7 ± 2.0 | 7.9 ± 2.2 | 8.8 ± 3.1 |
BMI, kg/m2 | 24.9 ± 2.4 | 25.1 ± 3.1 | 25.0 ± 2.9 | 25.3 ± 3.0 |
Hypertension, n (%) | 10(24.4) | 12(17.6) | 6(20.7) | 7(19.4) |
Hyperlipidemia, n (%) | 20(48.8) | 31(45.6) | 14(48.3) | 17(47.2) |
Current smoker, n (%) | 4(9.8) | 5(7.4) | 2(6.9) | 3(8.3) |
Current drinker, n (%) | 7(17.1) | 11(16.1) | 4(13.8) | 5(13.9) |
MMSE | 28.8 ± 1.0 | 25.4 ± 1.5 a | 25.3 ± 1.5 a | 23.2 ± 1.7 a, b, c |
MoCA | 26.8 ± 1.7 | 21.6 ± 1.9 a | 21.5 ± 1.6 a | 18.2 ± 3.4 a, b, c |
Total hippocampal volume, cm3 | 7.24 ± 0.73 | 6.39 ± 0.71 a | 5.99 ± 0.79 a, b | 5.11 ± 0.84 a, b, c |
AD signature cortical thickness, mm | 2.59 ± 0.18 | 2.55 ± 0.19 | 2.47 ± 0.13 a, b | 2.17 ± 0.33 a, b, c |
Total gray matter volume, cm3 | 612.3 ± 27.5 | 609.8 ± 22.4 | 607.5 ± 23.3 | 548.9 ± 24.8 a, b, c |
PVH, mm3 | 6119 ± 4464 | 8376 ± 6026 a | 9028 ± 6726 a | 11781 ± 8921 a, b |
DWMH, mm3 | 2871 ± 4756 | 3836 ± 4808 | 4367 ± 5179 | 6103 ± 6789 |
NDEs Aβ42, pg/ml | 3.17 ± 0.77 | 3.38 ± 0.84 | 3.78 ± 0.75 a, b | 4.15 ± 0.60 a, b, c |
NDEs T-tau, pg/ml | 163.5 ± 34.1 | 167.7 ± 30.0 | 184.7 ± 25.3 a, b | 207.2 ± 36.5 a, b, c |
NDEs P-T181-tau, pg/ml | 55.9 ± 10.2 | 57.3 ± 13.2 | 66.8 ± 16.5 a, b | 89.4 ± 22.0 a, b, c |
NDEs SNAP-25, pg/ml | 627.6 ± 152.0 | 593.0 ± 130.4 | 530.6 ± 131.5 a, b | 461.4 ± 116.9 a, b, c |
Abbreviations: CN cognitively normal, sMCI stable mild cognitive impairment, pMCI progressive mild cognitive impairment, AD Alzheimer's disease, BMI body mass index, MMSE Mini Mental State Examination, MoCA Montreal Cognitive Assessment, AD signature cortical thickness: cortical thickness in AD signature regions calculated as the average of cortical thickness in entorhinal, inferior temporal, middle temporal, and fusiform regions, PVH periventricular hyperintensities, DWMH deep white matter hyperintensities, NDEs neuronal-derived exosomes, SNAP-25, Synaptosomal - associated protein 25. |
a Significant at P < 0.05 versus CN; b Significant at P < 0.05 versus sMCI; c Significant at P < 0.05; Significant at P < 0.05 versus pMCI; d Significant at P < 0.05 versus AD. |
Plasma neuroexosomal mitochondrial protein levels in different diagnostic groups
Plasma neuroexosomal NDUFS3 (Fig. 1A) and SDHB (Fig. 1B) levels were significantly lower in T2DM patients with AD dementia (232.7 ± 63.4, 1360.7 ± 328.5, pg/ml) and pMCI (274.4 ± 78.6, 1536.7 ± 342.8, pg/ml) than in cognitively normal subjects (333.9 ± 96.7, 2050.4 ± 628.9, pg/ml) (P < 0.001 for both groups). Lower neuroexosomal NDUFS3 (Fig. 1A) and SDHB (Fig. 1B) levels were found in AD dementia (232.7 ± 63.4, 1360.7 ± 328.5, pg/ml) than in sMCI (319.9 ± 109.8 pg/ml, P < 0.001; 1824.7 ± 606.4 pg/ml, P < 0.001) and pMCI (274.4 ± 78.6 pg/ml, P < 0.05; 1536.7 ± 342.8 pg/ml, P < 0.05). We also found that plasma neuroexosomal NDUFS3 (Fig. 1A) and SDHB (Fig. 1B) levels were lower in pMCI (274.4 ± 78.6 pg/ml, 1536.7 ± 342.8 pg/ml) than in sMCI (319.9 ± 109.8 pg/ml, P < 0.05; 1824.7 ± 606.4 pg/ml, P < 0.05) subjects.
Associations between mitochondrial proteins and Aβ42 in plasma neuronal-derived exosomes
There were no significant associations between NDUFS3 and SDHB and Aβ42 in plasma neuronal-derived exosomes in CN (r = - 0.195, P = 0.222; r = - 0.259, P = 0.102) or sMCI (r = - 0.103, P = 0.405; r = - 0.225, P = 0.065) subjects (Fig. 2A, Fig. 2B). NDUFS3 and SDHB were negatively correlated with Aβ42 in pMCI (NDUF: r = - 0.462 P = 0.012; r = - 0.622, P < 0.001) and AD (r = - 0.527, P = 0.001; r = - 0.449, P = 0.006) patients (Fig. 2A, Fig. 2B)
Associations between mitochondrial proteins and tau biomarkers in plasma neuronal-derived exosomes
There were no significant associations between NDUFS3 and T-tau in plasma neuronal-derived exosomes in CN subjects (r = - 0.280, P = 0.076) (Fig. 3A). NDUFS3 was negatively correlated with P-181T-tau in CN subjects (r = - 0.321, P = 0.041) (Fig. 3C). There were no significant associations between SDHB and T-tau (r = - 0.289, P = 0.067) or P-181T-tau (r = - 0.277, P = 0.079) in plasma neuronal-derived exosomes in CN subjects (Fig. 3B, Fig. 3D). NDUFS3 and SDHB were not associated with T-tau in sMCI patients (Fig. 3A, Fig. 3B). There were significant associations of NDUFS3 and SDHB with P-181T-tau in plasma neuronal-derived exosomes in sMCI subjects (r = - 0.271, P = 0.026; r = - 0.276, P = 0.022) (Fig. 3C, Fig. 3D). NDUFS3 and SDHB were negatively correlated with T-tau (r = - 0.475, P = 0.009; r = - 0.448, P = 0.015) (Fig. 3A, Fig. 3B) and P-181T-tau (r = - 0.579, P = 0.001; r = - 0.448, P = 0.015) (Fig. 3C, Fig. 3D) in plasma neuronal-derived exosomes in pMCI patients. There were also significant associations of NDUFS3 and SDHB with T-tau (r = - 0.492, P = 0.002; r = - 0.583, P < 0.001) (Fig. 3A, Fig. 3B) and P-181T-tau (r = - 0.589, P < 0.001; r = - 0.699, P < 0.001) (Fig. 3C, Fig. 3D) in plasma neuronal-derived exosomes in AD patients.
Associations between mitochondrial proteins and SNAP-25 in plasma neuronal-derived exosomes
There were no significant associations between NDUFS3 and SDHB and SNAP-25 in plasma neuronal-derived exosomes in CN (r = 0.126, P = 0.433; r = 0.218, P = 0.170) (Fig. 4A, Fig. 4B) and sMCI (r = 0.205, P= 0.094; r = 0.214, P = 0.080) (Fig. 4A, Fig. 4B) subjects. Both NDUFS3 and SDHB were positively correlated with SNAP-25 in plasma neuronal-derived exosomes in pMCI (r = 0.614, P < 0.001; r = 0.633, P < 0.001) (Fig. 4A, Fig. 4B) and AD (r = 0.547, P = 0.001; r = 0.623, P < 0.001) (Fig. 4A, Fig. 4B) patients.
Plasma neuroexosomal mitochondrial proteins in relation to brain structure and WMH
The associations of mitochondrial proteins with baseline brain structure and WMH are shown in Table 2. Both NDUFS3 and SDHB were correlated with total hippocampal volumes in pMCI (β = 0.597, P = 0.011; β = 0.507, P = 0.028) and AD (β = 0.531, P = 0.001; β = 0.464, P = 0.004) patients. There were also significant associations of NDUFS3 and SDHB with AD signature cortical thickness in pMCI (β = 0.465, P = 0.014; β = 0.466, P = 0.010) and AD (β = 0.475, P = 0.003; β = 0.465, P = 0.003) patients. NDUFS3 and SDHB were only correlated with total gray matter volume in the AD group (β = 0.445, P = 0.005; β = 0.365, P = 0.025). Except for weak associations of NDUFS3 and SDHB with baseline PVH volumes in the AD group, neither baseline NDUFS3 nor SDHB levels were correlated with WMH (PVH and DWMH) volumes in the other groups.
Table 2
Analysis of association between mitochondrial proteins and baseline brain structure and WMH
| Total hippocampal volumes | AD signature cortical thickness | Total gray matter volume | PVH | DWMH |
β | P | β | P | β | P | β | P | β | P |
CN | | | | | | | | | | |
NDUFS3 | 0.150 | 0.356 | -0.049 | 0.761 | 0.008 | 0.963 | -0.222 | 0.159 | -0.095 | 0.554 |
SDHB | 0.143 | 0.380 | -0.158 | 0.323 | -0.101 | 0.541 | -0.226 | 0.151 | -0.156 | 0.327 |
sMCI | | | | | | | | | | |
NDUFS3 | 0.122 | 0.326 | 0.201 | 0.102 | 0.066 | 0.592 | -0.176 | 0.159 | -0.150 | 0.232 |
SDHB | 0.259 | 0.082 | 0.168 | 0.178 | -0.015 | 0.907 | -0.142 | 0.254 | -0.130 | 0.299 |
pMCI | | | | | | | | | | |
NDUFS3 | 0.597 | 0.011 | 0.465 | 0.014 | 0.353 | 0.065 | -0.122 | 0.549 | -0.143 | 0.469 |
SDHB | 0.507 | 0.028 | 0.466 | 0.010 | 0.344 | 0.062 | -0.135 | 0.490 | -0.242 | 0.199 |
AD | | | | | | | | | | |
NDUFS3 | 0.531 | 0.001 | 0.475 | 0.003 | 0.445 | 0.005 | -0.323 | 0.046 | -0.293 | 0.080 |
SDHB | 0.464 | 0.004 | 0.465 | 0.003 | 0.365 | 0.025 | -0.321 | 0.055 | -0.277 | 0.110 |
Abbreviations: CN cognitively normal, sMCI stable mild cognitive impairment, pMCI progressive mild cognitive impairment, AD Alzheimer's disease, NDUFS3 NADH ubiquinone oxidoreductase core subunit S3, SDHB Succinate dehydrogenase complex subunit B, AD signature cortical thickness: cortical thickness in AD signature regions calculated as the average of cortical thickness in entorhinal, inferior temporal, middle temporal, and fusiform regions, PVH periventricular hyperintensities, DWMH deep white matter hyperintensities. |
During the follow-up period, 29 MCI patients progressed to developing AD and were further subjected to brain MRI at the point of dementia conversion. Analysis of the correlation between mitochondrial proteins and changes in brain structure or WMH was performed. Lower baseline NDUFS3 and SDHB levels were correlated with volumetric loss in the total hippocampus (β = - 0.497, P = 0.007; β = - 0.524, P = 0.002), total gray matter (β = - 0.494, P = 0.008; β = - 0.431, P = 0.017), and reduced AD signature cortical thickness (β = - 0.302, P = 0.114; β = - 0.294, P = 0.111). There were no significant associations between baseline NDUFS3 or SDHB and increased PVH or DWMH volumes (data not listed).
Diagnostic power of plasma neuroexosomal mitochondrial proteins for pMCI and AD
The results obtained from the ROC curve analyses of the pMCI patients and CN subjects revealed that biomarkers in plasma neuronal-derived exosomes had lower diagnostic value for patients with pMCI (Table 3). Compared with Aβ42, T-tau, and SNAP-25, NDUFS3 and SDHB had almost the same range of diagnostic accuracy for AD (NDUFS3 vs. Aβ42, P = 0.594; NDUFS3 vs. T-tau, P = 0.862; NDUFS3 vs. SNAP-25, P = 0.724; SDHB vs. Aβ42, P = 0.975; SDHB vs. T-tau, P = 0.468; SDHB vs. SNAP-25, P = 0.340) (Fig. 5). Compared with P-181T-tau, SDHB also had almost the same range of diagnostic accuracy for AD (P = 0.114). However, P-181T-tau provided higher diagnostic accuracy than NDUFS3 for AD (P = 0.029) (Fig. 5).
Table 3
AUC of biomarkers in plasma neuronal-derived exosomes
| Aβ42 | T-tau | P-181T-tau | SNAP-25 | NDUFS3 | SDHB |
pMCI | 0.707(0.585-0.829) | 0.697(0.554-0.803) | 0.714(0.592-0.836) | 0.678(0.544-0.802) | 0.680(0.555-0.805) | 0.746(0.633-0.859) |
| (P = 0.003) | (P = 0.011) | (P = 0.002) | (P = 0.012) | (P = 0.011) | (P < 0.001) |
AD | 0831(0.742-0.920) | 0.790(0.691-0.889) | 0.907(0.839-0.975) | 0.779(0.678-0.880) | 0.801(0.705-0.896) | 0.833(0.745-0.920) |
| (P < 0.001) | (P < 0.001) | (P < 0.001) | (P < 0.001) | (P < 0.001) | (P < 0.001) |
Plasma neuroexosomal mitochondrial proteins in relation to cognition and future cognitive changes
There were no significant associations of NDUFS3 and SDHB in plasma neuronal-derived exosomes with baseline MoCA scores in CN (β = 0.072, P = 0.667; β = 0.143, P = 0.395) and sMCI (β = 0.135, P = 0.269; β = 0.203, P = 0.092) subjects. SDHB levels were correlated with baseline MoCA scores in pMCI (β = 0.448, P = 0.037) patients. There were no significant associations between NDUFS3 and baseline MoCA scores in pMCI (β = 0.260, P = 0.199) patients. Both NDUFS3 and SDHB were associated with baseline MoCA scores in AD patients (β = 0.431, P = 0.019; β = 0.418, P = 0.016).
Low NDUFS3 and SDHB levels in plasma neuronal-derived exosomes were correlated with a more rapid decrease in MoCA scores in pMCI during the clinical follow-up period (β = 0.481, P = 0.041; β = 0.474, P = 0.034).
Plasma neuroexosomal mitochondrial proteins predict conversion from MCI to AD
We analyzed whether plasma neuroexosomal mitochondrial proteins predicted conversion from MCI to AD. Cox proportional hazard regression analysis was performed for NDUFS3 and SDHB as continuous variables after adjusting for age and sex. Only SDHB significantly predicted conversion from MCI to AD. The hazard ratio (HR) was then calculated for SDHB as a dichotomous variable using the median values of SDHB as a cutoff (adjusting for age and sex). Subjects with low SDHB (HR 0.387, P = 0.018), corresponding to subjects whose SDHB values were ≤ 1645.8 pg/ml, progressed much more rapidly to AD than subjects with higher values (> 1645.8 pg/ml, corresponding to the higher median values of SDHB) (Fig. 6).