White matter alterations in Alzheimer's disease without concomitant pathologies

Abstract

Aims: Most individuals with AD neuropathological changes have co-morbidities which have an impact on the integrity of the WM. This study analyses oligodendrocyte and myelin markers in the frontal WM in a series of AD cases without clinical or pathological co-morbidities.

Methods: From a consecutive autopsy series, 206 cases had neuropathological changes of AD; among them, only 33 were AD without co-morbidities. WM alterations were first evaluated in coronal sections of the frontal lobe in every case. Then, RT-qPCR and immunohistochemistry were carried out in the frontal WM of AD cases without co-morbidities to analyse the expression of selected oligodendrocyte and myelin markers.

Results: WM demyelination was more marked in AD with co-morbidities when compared with AD cases without co-morbidities. Regarding the later, mRNA expression levels of MBP, PLP1, CNP, MAG, MAL, MOG and MOBP were preserved at stages I-II/0-A when compared with middle-aged (MA) individuals, but significantly decreased at stages III-IV/0-C. This was accompanied by reduced expression of NG2 and PDGFRA mRNA, reduced numbers of NG2-, Olig2- and HDAC2-immunoreactive cells and reduced glucose transporter immunoreactivity. Partial recovery of some of these markers occurred at stages V-VI/B-C.

Conclusions: The present observations demonstrate that co-morbidities have an impact on WM integrity in the elderly and in AD, and that early alterations in oligodendrocytes and transcription of genes linked to myelin proteins in WM occur in AD cases without co-morbidities. These are followed by partial recovery attempts at advanced stages. These observations suggest that oligodendrocytopathy is part of AD.

Keywords: Alzheimer disease; co-morbidities; myelin; oligodendrocytes; white matter.

Figure 1

Representative formalin‐fixed, paraffin‐embedded, de‐waxed coronal sections of the frontal cortex at the level of the head of the caudate and putamen, stained with Klüver–Barrera. (A) AD stage V/C, the absence of co‐morbidities (M, 82y); (B) ADIII–IV/A presenting with a frontal infarct (M, 78y); (C) Patient categorized as mixed dementia suffering from HTA and extensive WM hyper‐lucencies, AD stage III–IV/A, LBD stage 3 (M, 75y); (D) Patient with chronic respiratory insufficiency and terminal hypoxia, AD pathology stage II/0 (M, 74y); (E) Patient with chronic respiratory failure, abnormal behaviour of nondetermined origin and AD‐ pathology stage III/0 (M, 54y); (F) Patient with cognitive impairment, focal WM hyper‐lucencies, lacunar infarcts, HTA and AD stage V/C (M, 67y); (G) Patient with cognitive impairment, HTA, WM hyper‐lucencies and AD pathology stage II/0 (M, 40y); (H) Patient with severe cognitive impairment, type II diabetes, hyperlipidaemia, obesity, HTA, renal failure, argyrophilic grain disease stage II and AD pathology III/A (M, 76y); (I) Patient with hepatic encephalopathy and AD pathology stage III/A (M, 58y); (J) Patient with no neurological symptoms, and the absence of clinical and pathological co‐morbidities, categorized as AD stage III/A (M, 69y); (K) Patient with mild cognitive impairment, WM hyper‐lucencies, HTA and AD pathology stage III/B (M, 72y); (L) Patient with long‐lasting dementia, the absence of risk factors of cerebral circulatory disturbance, and affected by ADVI/C, argyrophilic grain disease stage II and TDP‐43 proteinopathy (M, 76). The figure makes it evident that there is variable involvement of the WM in association with distinct cerebral and systemic disorders concomitant with AD pathology. M: man. Compare the variability of WM involvement in cases B, C, E, H, I and K categorized as AD with co‐morbidities (AD‐Co) with J categorized as AD without co‐morbidities, all of them stage ADIII–IV of Braak and Braak. Cases D and G, classified as AD‐Co stage II, also show decreased staining of the centrum semi‐ovale when compared with stage III of AD without co‐morbidities (J). Differences are also observed between AD without co‐morbidities stage V (A) in comparison with AD‐Co stages V and VI (F and L). Note that all cases in the figure correspond to males to avoid gender bias).

Figure 2

Densitometric values of myelin sheet phospholipids as revealed in Klüver–Barrera‐stained sections of the centrum semi‐ovale at the level of the head of the caudate and putamen in MA, AD with co‐morbidities (AD‐Co), and cases of AD without co‐morbidities (AD). Values are expressed as arbitrary units per area. (A) The area of densitometric studies is indicated by the circle; Klüver–Barrera staining in an MA case. Note that the area is separated from the periventricular white matter and the subcortical U‐fibres. (B) Significant decrease in AD without co‐morbidities (n = 33) and AD‐Co (n = 173) is seen when compared with MA (n = 20). The intensity of myelin staining is significantly lower in AD‐Co when compared with AD cases. (C) No significant differences, but a tendency to reduced myelin intensity is seen in AD without co‐morbidities at stages V–VI/B–C (ADV–VI) when compared with AD at stages I–II/0‐A (ADI–II) and AD at stages III–IV/0‐C (ADIII–IV). ADI–II/0‐A, n = 9; ADIII–IV/0–C, n = 8; ADV–VI/B–C, n = 16. One‐way anova and post hoc Tukey, ***P < 0.001 AD and AD‐Co compared with MA; ^## P < 0.001: AD‐Co compared with AD without co‐morbidities.

Figure 3

mRNA expression of selected oligodendrocyte‐ and myelin‐related genes in the frontal white matter of MA, AD without co‐morbidities stages I–II/0‐A (ADI–II), III–IV/0‐C (III–VI) and V–VI (B, C). Abbreviations may be seen in Table 2. One‐way analysis of variance (anova) followed by Tukey posttest or Kruskal–Wallis test followed by Dunn’s post hoc test when required using the SPSS software; *P < 0.05, **P < 0.01, ***P < 0.001 vs. MA; ^# P < 0.05, ^## P < 0.01 vs. ADI–II; ^$ P < 0.05 vs. ADIII–IV and ADV–VI (see Methods for statistical studies).

Figure 4

Immunohistochemistry to cellular markers NG2, Olig2, HDAC2 and SLC2A1:GLUT1 in MA individuals (A, E, I, M), and in cases with AD without co‐morbidities at stages ADI–II/0‐A (ADI–II) (B, F, J, N), ADIII–IV/0–C (ADIII–IV) (C, G, K, O) and ADV–VI/B–C (ADV–VI) (D, H, L, P). Decreased numbers of NG2‐, Olig2‐ and HDAC2‐immunoreactive cells are observed at middle, and particularly, advanced stages of AD. Large hyperchromatic Olig2‐positive cells are also observed in ADV–VI. GLUT1 immunoreactivity is manifested as a fine uniform meshwork in the neuropil which is progressively disrupted into patches of variable immunoreactivity with disease progression. Paraffin sections, slightly counterstained with haematoxylin; NG2, HDAC2, bar = 50 µm; Olig2 and SLC2A1, bar = 45 µm. Insert in MA NG2 is at greater magnification to show small positive granules characteristic of NG2 immunoreactivity. Insert in Olig2 ADV–VI shows a representative large hyperchromatic Olig2‐immunoreactive cell; these cells are commonly present in ADV–VI.

Figure 5

Immunohistochemistry to myelin markers PLP1, CNPase and MBP in the centrum semi‐ovale of the frontal lobe in MA (A, E, I), and in cases with AD without co‐morbidities at stages ADI–II/0‐A (ADI–II) (B, F, J), ADIII–IV/0‐C (ADIII–IV) (C, G, K) and ADV–VI/B–C (ADV–VI) (D, H, L). Representative images show reduced immunoreactivity with disease progression, and small PLP1‐ and CNPase‐immunoreactive dots in ADV–VI. Paraffin sections, slightly counterstained with haematoxylin; bar = 50 µm.

Figure 6

Quantitative study of NG2‐, Olig2‐ and HDAC2‐immunoreactive cells in the frontal WM per area of AD cases without co‐morbidities (0.038 mm² for NG2‐ and HDAC2‐immunoreactive cells, and 0.126 mm² for Olig2‐positive cells; see Methods). The number of positive cells decreases with increasing stages of AD pathology. MA, n = 10; ADI–II/0‐A, n = 9; ADIII–IV/0‐C, n = 8; ADV–VI/B–C, n = 16. One‐way anova and post hoc Tukey; *: MA vs. ADIII–IV/0‐C (ADIII–IV) or ADV‐VI/B–C (ADV–VI); #: ADI–II/0‐A (ADI–II) vs. ADIII–IV or ADV–VI; $: ADIII–IV vs. ADV–VI; significance level set at *^{, #} P < 0.05, ** P < 0.01 and ***, ^###, ^$$$ P < 0.001.