Mixed Brain Pathology Is the Most Common Cause of Cognitive Impairment in the Elderly

Abstract

Background:

Systemic diseases, diabetes mellitus (DM), and cardiovascular disease (CaVD) have been suggested being risk factors for cognitive impairment (CI) and/or influence Alzheimer’s disease neuropathologic change (ADNC).

Objective:

The purpose was to assess the type and the extent of neuropathological alterations in the brain and to assess whether brain pathology was associated with CaVD or DM related alterations in peripheral organs, i.e., vessels, heart, and kidney.

Methods:

119 subjects, 15% with DM and 24% with CI, age range 80 to 89 years, were chosen and neuropathological alterations were assessed applying immunohistochemistry.

Results:

Hyperphosphorylated τ (HPτ) was seen in 99%, amyloid-β (Aβ) in 71%, transactive DNA binding protein 43 (TDP43) in 62%, and α-synuclein (αS) in 21% of the subjects. Primary age related tauopathy was diagnosed in 29% (more common in females), limbic predominant age-related TDP encephalopathy in 4% (14% of subjects with CI), and dementia with Lewy bodies in 3% (14% of subjects with CI) of the subjects. High/intermediate level of ADNC was seen in 47% and the extent of HPτ increased with age. The extent of ADNC was not associated with the extent of pathology observed in peripheral organs, i.e., DM or CaVD. Contrary, brain alterations such as pTDP43 and cerebrovascular lesions (CeVL) were influenced by DM, and CeVL correlated significantly with the extent of vessel pathology.

Conclusion:

In most (66%) subjects with CI, the cause of impairment was “mixed pathology”, i.e., ADNC combined with TDP43, αS, or vascular brain lesions. Furthermore, our results suggest that systemic diseases, DM and CaVD, are risk factors for CI but not related to ADNC.

Keywords

Aging amyloid-β cardiac pathology cerebrovascular lesions nephrosclerosis transactive DNA binding protein 43

INTRODUCTION

Cardiovascular disease (CaVD) has been implicated as being a cause of age-related cognitive impairment (CI), particularly for Alzheimer’s disease (AD) [1 –4]. Some studies have even suggested that a causative association exists between Alzheimer’s disease neuropathologic changes (ADNC) and CaVD [5 –8]. Neuropathologically, AD is defined as a neurodegenerative disease with a progressive accumulation of altered proteins in the brain, i.e., amyloid-β (Aβ) and hyperphosphorylated tau (HPτ) [9, 10]. These alterations are observed in defined neuroanatomical brain regions and display an orderly progression within the brain [9, 10]. A sufficient extent of these altered proteins, based on a regional distribution, i.e., high level of ADNC, is required for a definite neuropathologic diagnosis of AD [11, 12]. Noteworthy, since the use of immunohistochemical (IHC) technique while assessing postmortem (PM) brain, it has become evident that the clinical syndrome with AD characteristics is preceded by a lengthy asymptomatic phase with ADNC [13, 14]. The progressive accumulation of ADNC from low, intermediate, to finally, high level is readily assessed PM [15, 16]. However, it is rather difficult to assess the clinical signs observed at the low and/or intermediate levels of ADNC. This is particularly difficult, as in the elderly population ADNC is frequently observed in association with other, concomitant, pathologies [17]. Several reports have suggested that concomitant pathologies when seen with low and intermediate level of ADNC might be causative regarding the clinically observed CI [18 –20]. It is well known today that many subjects with sporadic dementia disorder with a clinical phenotype of AD display, not only ADNC at a neuropathological assessment but also other age-related protein alterations such as concomitant accumulation of phosphorylated transactive DNA binding protein 43 (pTDP43) and/or phosphorylated α-synuclein (αS) [17 , 21–24]. In line with the assessment of the extent of ADNC, consensus agreement is available regarding the assessment of the extent of αS and pTDP43 when implementing the IHC technique and investigating defined neuroanatomical regions [21 –26]. Thus, a reliable assessment of the extent of ADNC, αS, and pTDP43 can indeed be obtained.

When a demented subject displays cerebral vascular lesions (CeVL), the diagnosis is vascular dementia (VaD) or vascular cognitive impairment (VCI) [28, 29]. The former, VaD, refers to a subject with CI where the PM neuropathological investigation reveals only CeVL, whereas in VCI, CeVL are observed concomitant with other neurodegenerative alterations [30, 31]. Currently, there is no consensus regarding the type or extent of CeVL that has to be seen in the brain for a diagnosis of VaD/VCI [32]. Systematic assessment of CeVL in subjects with VaD has indicated that cribriform change and micro-infarcts in deep grey/white matter are associated with CI [33, 34]. Noteworthy, current neuropathological diagnosis of VaD or VCI is still based on a subjective assessment of various CeVL (infarcts, white matter rarefaction, alterations in the vessel walls) and exclusion of widespread ADNC, pTDP43, or αS pathology. The stepwise progression of ADNC, the recently defined entities of Primary Age Related Tauopathy (PART), the Aging Related Tau Astrogliopathy (ARTAG), and the Limbic Predominant Aging related TDP encephalopathy (LATE) certainly challenge the existence of pure VaD [24 , 36]. The pure VaD, i.e., a case lacking altered proteins when reliable IHC technique is implemented, is probably extremely uncommon, whereas VCI, i.e., a case with CeVL that contributes to other pathologies and thus alters the severity and the phenotype of the dementia syndrome, might be underestimated [20, 30].

The association between a number of vascular risk factors such as hypertension (HT), general atherosclerosis, CaVD, and the clinical manifestation of AD syndrome is primarily supported by epidemiological studies [1, 37]. Contrary to the above, a direct causative association between vascular risk factors and ADNC is controversial [18 , 37–39]. The assessment of a causative association between ADNC and vascular risk factors in human setting is challenging. First, many subjects with a clinical phenotype of AD display not only ADNC at a neuropathological assessment but also other concomitant alterations, including CeVL as mentioned above. Secondly, reliable assessment of the influence of a systemic disease, often not one but several, might be difficult. CeVL can be caused by HT, CaVD, diabetes mellitus (DM), or metabolic syndrome or all the above, i.e., cumulative effect of these diseases on the brain tissues. Furthermore, in 2016, it was reported that a large number of adults in the U.S. have underdiagnosed type 2 DM, and as many as 5 to 10% of the European population display impaired glucose metabolism indicating that the diagnosis of DM is ambiguous [40, 41]. Moreover, large meta-analysis has indicated that DM and metabolic syndrome are associated with increased incidence of dementia but only when co-existing with mild CI [37]. Likewise, a positive association between dementia and history of stroke has been noted, whereas HT, heart disease, dyslipidemia, and DM have been observed as being positively associated with VaD/VCI but not with AD [39]. Noteworthy, in the industrial world where various more or less efficient treatment strategies are implemented, it might be rather difficult, if not impossible, to reliably estimate the impact of common systemic diseases of the heart, kidney, or brain tissue as listed above. Thus, it is not surprising that randomized controlled trials that target the major CaVD risk factors have generally failed to be efficacious in prevention of AD [42].

Nevertheless, the eventual causative association between ADNC and CaVD needs to be assessed. It is of importance to recognize all currently acknowledged brain alterations significant for CI (ADNC, pTDP43, αS, and cerebrovascular lesion) and assess their association with CaVD presumed being of significance. For a reliable assessment of CaVD, instead of registering the clinical diagnosis and eventual treatment strategies, alterations caused by CaVD and observed in peripheral organs such as the brain, kidney, and heart can be assessed. HT and/or atherosclerosis are associated with histologically assessable chronic changes in the kidney, i.e., nephrosclerosis, the heart muscle displays myocardial hypertrophy and infarcts, and the brain displays all pathologies discussed above [43, 44]. With this approach, the deleterious influence of a disease on the target organ is assessed independent of the duration of the disease or of various treatment strategies. It should also be kept in mind that the latter is influenced by the level of stringency with which the patient follows the treatment recommendations. In line with the above, a subject with a clinical diagnosis of HT and/or CaVD, under efficient pharmacological treatment, probably displays less severe alterations in the organ of interest, i.e., mild nephrosclerosis, lack of myocardial hypertrophy, and/or infarcts as well as displays sparse alterations in the brain when compared with a less efficiently treated counterpart.

In this study, the pathological alterations seen in the organs of interest in humans upon death, i.e., the vessels, the brain, the heart, and the kidney were assessed. The brain was chosen as the organ of interest regarding CI; the vessels, the kidney, and the heart were chosen as they are known to display assessable lesions related to clinical diagnoses of HT and CaVD.

MATERIAL AND METHODS

This study was carried out on PM obtained tissue, and the study was approved by the local ethical committee (Dnr 2011/286), updated in 2015.

The autopsy was carried out following standard procedure. The PM delay was noted, the brain was removed, weighed and placed in 4% buffered formalin. After 2 to 5 days fixation, the grossing was carried out, the severity of arteriosclerosis was registered (none, mild, moderate/severe), and macroscopic lesions to be seen were noted on coronal sections. A standard setting of brain tissue was sampled (neuroanatomical regions listed in Supplementary Table 1) and placed in commercial mega cassettes for further fixation in 4% buffered formalin. After an additional 2 weeks fixation, the automatic paraffin embedding procedure was initiated. Parallel to the removal of the brain, gross assessment of the corpse was carried out. The grade of the general and coronal athero-/arteriosclerosis was estimated (none, mild, moderate/severe). All organs including the kidneys and the heart were weighed and inspected for gross pathology; thereafter, representative samples were taken and placed in commercial cassettes, fixed in formalin, and then embedded for histology. The autopsy generated a statement regarding the final cause of death. For this study, stained sections from the brain, kidney, and heart as listed in Supplementary Table 1 were histologically assessed.

Overall, during a period of 4 years, a total of 440 complete autopsies (240 males, 200 females, mean age 74±0.6, 20% with CI), including a neuropathological investigation had been carried out. For this study, only subjects within the age range of 80 to 89 years at death were included. The relatively high and limited age range was chosen as all pathologies assessed in this study are age related, and the extent of alterations is known to increase with the aging of the subject. There were 134 subjects (68 males and 66 females, 29% with CI) that fulfilled the selection criteria. Thereafter, subjects with a neuropathologically verified primary tauopathy (2 cases) or frontotemporal lobar degeneration (5 subjects) were excluded. Due to the insufficient quality of samples obtained from the kidney and the heart, an additional eight cases had to be excluded, three of which had displayed CI during life. Thus, this study included 119 cases, 61 females and 58 males, of which 29 (24%) subjects had displayed CI during life.

Tissue samples assessed and stains used are summarized in Supplementary Table 1. All stains were carried out in automatic systems. The section thickness of the peripheral organ samples was 4μm and the section thickness of the brain tissue samples was 7μm. The antibodies and methods applied for the IHC stains for visualization of the misfolded proteins are listed in Supplementary Table 2.

In the brain tissue, vascular alterations were assessed in hematoxylin and eosin (H&E) stained sections in magnification ×20–×100. Microscopic infarcts, disregarding the size or type, were registered for each H&E stained section. In the sections with striatum, cribriform changes and lacunar infarcts were registered. Cerebral amyloid angiopathy (CAA) was registered in IHC stained sections, applying antibody directed to Aβ protein (Supplementary Table 2). ARTAG was registered as seen or not seen in the section of the basal forebrain while applying the antibody directed to HPτ protein (Supplementary Table 2). Staging of observed protein alterations seen in the IHC stained sections was based on the current consensus criteria, i.e., Braak stage for HPτ, Thal phase for Aβ, Josephs stage for pTDP43, and Braak stage for αS [9 , 26].

A score for the extent of vessel changes (ArT) was assigned based on three parameters, i.e., the extent (no, mild, moderate/severe) of general atherosclerosis seen in the main aorta and its larger tributaries, coronary arteriosclerosis, and arteriosclerosis in the circulus Willisii. The ArT score ranged from 1 to 6 (Table 1). A score for CeVL was assigned based on four parameters, i.e., whether a gross infarct was seen with the naked eye, the number of microscopic infarcts seen in the H&E stained brain sections, observation of cribriform changes, and/or lacunar infarcts in the central grey matter. The CeVL score ranged from 0 to 7 (Table 1). Kidney sections were assessed in the H&E stained section, and a score of the severity of nephrosclerosis (NpS) was assigned for each case. The NpS score is a modification from what was recommended in 2017 by Sethi and colleagues while assessing chronic changes in the kidney [45]. The modified score is based on three parameters, i.e., the extent of glomerulosclerosis, interstitial fibrosis, and arteriosclerosis. The assessment of tubular atrophy included in the original assessment recommendations by Sethi and colleagues was excluded, being unreliable in the PM material [45]. The obtained score ranged from 0 to 5 (Table 1). A score of cardiac pathology (CorP) was based on two parameters, i.e., the weight of the heart muscle and whether a tissue alteration, infarct was noted at gross examination (Table 1). When in doubt regarding the gross findings of an infarct, a microscopic assessment was carried out of the H&E stained heart sections.

Table 1

Scoring the severity of lesions assessed postmortem in the vessels, brain, heart, and kidney

Alteration to be assessed	Extent of an alteration			Sum of severity (A,B,C) of alterations/Score
	A = 0	B = 1	C = 2	The extent of alterations in the vessels, ArT (range 1–6)
General atherosclerosis^*	no	Mild	moderate/severe	mild 1 - 2
Coronary arteriosclerosis^*	no	Mild	moderate/severe	moderate 3 - 4
Arteriosclerosis in circulus Willisii*	no	mild	moderate/severe	severe ≥5
				The extent of cerebral vascular lesions, CeVL (range 0–7)
Gross infarct^#			Seen	none 0
Micro infarcts¹	no	in one slide	in >than 1 slide	mild 1
Cribriform change in central grey¹		seen		moderate 2
Lacunar infarct in central grey¹			Seen	severe ≥3
				The extent of alterations in the kidney, NpS (range 0–5)
Glomerulosclerosis¹	mild	moderate	Severe	none 0
Interstitial fibrosis¹	mild	moderate	Severe	mild 1
Arteriosclerosis¹		intima thicker than media		moderate 2
				severe ≥3
				The extent of alterations in the heart, CorP (range 0–2)
Heart weight		≥500 grams		none 0
Infarct		Seen		mild 1
				severe 2

^*seen with the naked eye in main aorta and its larger tributaries, in the heart vessels or in the circulus Willisii (mild - focal plagues, moderate/severe - multifocal with or without lumen obliteration); ^#seen with the naked eye; ¹seen in hematoxylin eosin stained sections.

For statistics, IBM SPSS was used applying non-parametric tests. Descriptive statistics was given as mean±Standard Error (m±SE) of means. For statistical difference between the studied groups, Mann-Whitney-U (MWU) test, Fisher’s test for contingency of categorical data, and Spearman correlation test for correlation between the studied variables were applied.

RESULTS

The distribution of CI and DM was even between males and females and the age at death did not differ significantly between the genders (Table 2). The mean weight of the brain and kidney was significantly higher in males, whereas the mean PM delay was significantly higher in females (Table 2). When comparing subjects with and without CI, only the heart weight differed significantly, being higher in subjects without CI. The most common cause of death was cardiovascular 64% (males/females 38/38), followed by infectious disease 27% (males/females 17/15), and neoplasia 9% (males/females 3/8).

The incidence of pathologies in the brain of the subjects did not differ based on the gender (Table 3). Noteworthy, however, some of the alterations that are part of the CeVL score differed between the genders. Gross brain infarcts that were observed with the naked eye were more common in females when compared to males, and cribriform changes with lacunar infarcts in the central grey observed with light microscopy were more common (Fisher’s exact test, p = 0.017) in males when compared with females. HPτ and αS were observed in the brain as frequently in subjects with or without CI, whereas alterations such as Aβ, pTDP43, CAA, and ARTAG were significantly more common in subjects with CI (Table 3). Contrary to the above, CeVL was more common in subjects without CI.

Table 2

Demographics, groups based on gender or cognitive impairment (CI), statistics when comparing males/females or subjects with/without CI

	Value	All	Female	Male	Statistics
Number		119	61	58
Postmortem delay, h	m±SE	101.2±4.9	110 ± 7	92 ± 7	MWU/p = 0.031
Age at death, y	m±SE	84.1±0.2	84.4±0.4	83.9±0.3	MWU/ns
Brain weight, g	m±SE	1319±13	1241 ± 15	1401 ± 16	MWU/p = 0.000
Kidney weight, g	m±SE	233±5	212 ± 6	255 ± 7	MWU/p = 0.000
Heart weight, g	m±SE	459±11	439±13	480±16	MWU/ns
With CI	(percent)	29 (24)	14 (23)	15 (26)	FE/ns
With diabetes mellitus	(percent)	18 (15)	10 (16)	8 (14)	FE/ns
			without CI	with CI
Number		119	90	29
Female/male			47/43	14/15
Post mortem delay, h	m±SE		100±5	106±11	MWU/ns
Age at death, y	m±SE		84.0±0.3	84.5±0.5	MWU/ns
Brain weight, g	m±SE		1325±16	1301±24	MWU/ns
Kidney weight, g	m±SE		239±6	216±11	MWU/ns
Heart weight, g	m±SE		470 ± 12	424 ± 20	MWU/p = 0.02
With diabetes mellitus	(percent)		14 (16)	4 (14)	FE/ns

m±SE, mean±standard error of mean; MWU, Mann Whitney U tests; FE, Fisher’s exact test.

Table 3

Incidence of the assessed alteration in 119 subjects, grouped based on gender or cognitive impairment (CI)

Organ	Type of alteration	n (%)	Female n (%)	Male n (%)	Statistics Fisher’s exact
Brain	All	119	61 (51)	58 (49)
	with hyperphosphorylated τ	118 (99)	61 (100)	57 (98)	ns
	with amyloid-β	85 (71)	40 (66)	45 (78)	ns
	with pTDP43	74 (62)	37 (61)	37 (64)	ns
	with α-synuclein	25 (21)	12 (20)	13 (22)	ns
	with CAA	32 (27)	14 (23)	18 (31)	ns
	with ARTAG	56 (47)	28 (46)	28 (48)	ns
	with severe CeVL	9 (8)	6 (10)	3 (5)	ns
Vessels	with severe ArT	71(60)	36 (59)	35 (60)	ns
Kidney	with severe NpS	46 (39)	25 (41)	21 (36)	ns
Heart	with severe CoP	19 (16)	6 (10)	13 (22)	0.05
			without CI n (%)	with CI n (%)
Brain	All		90 (76)	29 (24)
	with hyperphosphorylated τ	89 (99)	29 (100)	ns
	with amyloid-β	56 (62)	29 (100)	0.000
	with pTDP43	49 (54)	25 (86)	0.001
	with α synuclein	20 (22)	5 (17)	ns
	with CAA	17 (19)	15 (52)	0.001
	with ARTAG	37 (31)	19 (66)	0.019
	with severe CeVL	8 (9)	1 (3)	ns
Vessels	with severe ArT	56 (62)	15 (52)	ns
Kidney	with severe NpS	32 (36)	14 (48)	ns
Heart	with severe CoP	18 (20)	1 (3)	0.025

pTDP43, phosphorylated transactive DNA binding protein 43; CAA, cerebral amyloid angiopathy; ARTAG, Aging Related Tau Astrogliopathy; for CeVL, cerebral vascular lesion; ArT, vessel pathology; NpS, nephroslerosis and CoP, cardiac pathology, see Table 1.

In Table 4, the distribution of the extent of pathologies is listed in relation to gender or CI. The number of subjects with a moderate to severe extent of misfolded proteins such as HPτ, Aβ, and pTDP43 was significantly higher in subjects with CI when compared with subjects without CI, whereas gender lacked any significant impact on these alterations. Contrary to the above, moderate and/or severe CeVL and severe NpS were significantly more frequent in males when compared to females (Table 4).

Table 4

Distribution of the severities of the assessed alterations in relation to gender and cognitive impairment (CI)

Organ	Lesion type	Female n (%)	Severity (%)				Male n (%)	Severity (%)				Statistics Fisher’s exact
			None	Mild	Moderate	Severe		None	Mild	Moderate	Severe
Brain	all	61 (51)					58 (49)
	HPτ	61 (100)		29 (48)	27 (44)	5 (8)	57 (98)	1 (2)	27 (45)	25 (43)	5 (9)	ns
	Aβ	40 (66)	21 (34)	12 (20)	13 (21)	15 (25)	45 (79)	13 (22)	16 (28)	11 (19)	18 (31)	ns
	pTDP43	37 (61)	24 (39)	11 (18)	20 (33)	6 (10)	37 (64)	21 (36)	13 (23)	20 (34)	4 (7)	ns
	αS	12 (20)	49 (80)	1 (2)	7 (11)	4 (7)	13 (22)	45 (78)		10 (17)	3 (5)	ns
	CeVL	32 (52)	29 (48)	17 (28)	9 (15)	6 (10)	35 (60)	23 (40)	9 (15)	23 (40)	3 (5)	0.017
Vessels	ArT	61 (100)		1 (2)	24 (39)	36 (59)	58 (100)			23 (40)	35 (65)	ns
Kidney	NpS	49 (80)	12 (20)	10 (16)	14 (23)	25 (41)	49 (84)	9 (16)	12 (20)	16 (28)	21 (36)	0.054
Heart	CorP	33 (54)	28 (46)	27 (44)		6 (10)	42 (72)	16 (28)	29 (50)		13 (22)	ns
		without CI					with CI
		n (%)					n (%)
Brain	all	90 (76)					29 (24)
	HP τ	89 (99)	1 (1)	56 (62)	33 (37)		29 (100)			19 (66)	10 (34)	0.000
	Aβ	56 (62)	34 (38)	23 (26)	20 (22)	13 (14)	29 (100)		5 (17)	4 (14)	20 (69)	0.000
	pTDP43	49 (54)	41 (45)	16 (18)	26 (29)	7 (8)	25 (86)	4 (14)	8 (28)	14 (48)	3 (10)	0.023
	αS	20 (22)	70 (78)	1 (1)	14 (16)	5 (5)	5 (17)	24 (83)		3 (10)	2 (7)	ns
	CeVL	56 (52)	34 (38)	21 (23)	27 (30)	8 (9)	11 (38)	18 (62)	5 (17)	5 (17)	1 (4)	ns
Vessels	ArT	90 (100)		1 (1)	33 (37)	56 (62)	29 (100)			14 (48)	15 (52)	ns
Kidney	NpS	72 (80)	18 (20)	16 (18)	24 (27)	32 (36)	26 (90)	3 (10)	6 (21)	6 (21)	14 (48)	ns
Heart	CoP	59 (66)	31 (34)	41 (46)		18 (20)	16 (55)	13 (45)	15 (52)		1 (3)	ns

CI, cognitive impairment; HPτ, hyperphosphorylated τ (Braak stage [9] 0/none, a,b,I,II/mild; III,IV/ moderate, ≥V/ severe); Aβ, amyloid-β (Thal phase [10] 0/none, 1/mild, 2,3/moderate, ≥4 /severe); αS, α synuclein (Braak stage [26] 0/none, 1,2/mild, 3,4/moderate; ≥5/severe); pTDP43, phosphorylated transactive DNA binding protein 43 (Josephs stage [22] 0/none, 1/mild, 2,3/moderate, ≥4/severe); for severity of ArT, vessel pathology; CeVL, cerebral vascular lesions; NpS, nephrosclerosis; CorP, cardiac pathology; see Table 1.

Gender did not influence the extent (m±SE) of assessed brain pathologies (Table 5). Contrary to the above, the CorP was significantly more severe in males when compared to females. In subjects with CI, a significantly higher extent of HPτ, Aβ, and pTDP43 was observed when compared to neurologically unimpaired subjects. In contrast, the extent of CeVL was significantly higher in subjects without CI when compared to subjects with CI (Table 5). The extent of HPτ was significantly lower (MWU test, p = 0.000) in subjects with DM (m±SE, 1.94±1.11) when compared with subjects without DM (m±SE, 2.68±1.39), whereas the extent of CeVL and ArT was significantly higher (MWU test, p = 0.01 respective 0.05) in subjects with DM (m±SE, 1.56±1.29 respective 5.22±0.88) when compared with those without DM (m±SE, 0.94±1.08 respective 4.55±0.95).

Table 5

Gender, cognitive impairment (CI), and the severity of pathological alterations seen in the brain, vessels, heart, and kidney

	Value	Range	Mean±SE	Female	Male	Without CI	With CI
Number			119	61	58	90	29
Hyperphosphorylated τ	Braak stage⁹	0–6	2.57±0.13	2.56±0.18	2.59±0.18	2.03 ± 0.11³	4.24 ± 0.15³
Amyloid-β	Thal phase¹⁰	0–5	1.98±0.16	1.87±0.23	2.10±0.22	1.49 ± 0.16³	3.52 ± 0.24³
pTDP43 pathology	Josephs stage²²	0–5	1.39±0.13	1.41±0.19	1.38±0.18	1.21 ± 0.15³	1.97 ± 0.26³
α synuclein	Braak stage²⁶	0–6	0.87±0.16	0.79±0.21	0.97±0.24	0.90±0.18	0.79±0.33
Cerebral Vascular Lesions (CeVL)	see Table 1	0–4	1.03±0.10	0.93±0.15	1.14±0.14	1.16 ± 0.12¹	0.66 ± 0.19¹
Vascular pathology (ArT)	see Table 1	2–6	4.66±0.09	4.66±0.13	4.66±0.12	4.72±0.10	4.45±0.18
Nefrosclerosis (NpS)	see Table 1	0–4	1.93±0.11	1.87±0.15	2.00±0.17	1.86±0.13	2.17±0.22
Cardiac pathology (CorP)	see Table 1	0–2	0.79±0.06	0.64 ± 0.08²	0.95 ± 0.09²	0.86±0.08	0.59±0.11

pTDP43, phosphorylated transactive DNA binding protein 43; m±SE, mean±standard error of mean; Significant differences Mann Whitney U tests; ¹p = 0.05, ²p = 0.01, ³p = 0.000.

All significant correlations between age and the assessed tissue alterations listed in Table 5 are summarized in Table 6. Furthermore, the influence of gender and DM on the correlations was investigated. The age (80 to 89 years) influenced significantly the extent of HPτ and pTDP43. The extent of HPτ and Aβ displayed a significant strong correlation and was not influenced by gender or DM. The pTDP43 displayed a strong significant correlation with both HPτ and Aβ. A correlation between pTDP43/HPτ and TDP43/Aβ was lacking in subjects with DM. The Aβ/αS displayed a significant positive correlation but only in males or in subjects without DM. A negative significant correlation was observed between CeVL/HPτ, CeVL/Aβ, and CeVL/pTDP43. A significant positive correlation was observed between HPτ/ NpS but only in subjects without DM. In line with the above, a significant positive correlation was observed between αS/NpS but only in subjects with DM. Contrary to the above, the correlation was negative, robust, and significant between Aβ/NpS but only in subjects with DM. A robust significant correlation was observed between ArT, CeVL, and CorP.

Table 6

Spearman’ rho (r) correlations when significance 0.05¹, 0.01²

	All	M/F	–DM/+DM
Number	119	58/61	101/18
Hyperphosphorylated (HP) τ/age	0.22¹
pTDP43/age	0.22¹	0.31¹/ns	ns/0.50¹
HPτ/pTDP43	0.40²	0.41²/0.40²	0.38²/ns
HPτ/amyloid-β (Aβ)	0.59²	0.52²/0.66²	0.58²/0.65²
Aβ/pTDP43	0.44²	0.51²/0.39²	0.47²/ns
Aβ/α Synuclein (α)		0.27¹/ns	0.23¹/ns
HPτ/Cerebral Vascular Lesions (CeVL)	–0.19¹
HPτ/Nephrosclerosis (NpS)			0.24¹/ns
pTDP43/CeVL	–0.26²	ns / –0.33²	ns/–0.64²
Aβ/CeVL	–0.26²	–0.28¹/–0.28¹	–0.21¹/ns¹
Aβ/vessel pathology (ArT)	–0.23¹	ns / –0.25¹	–0.22¹/ns
Aβ/NpS			ns/–0.63²
αS/NpS			ns/0.52¹
ArT/CeVL	0.52²	0.50²/0.57²	0.50²/0.57¹
ArT/Cardiac Pathology (CorP)	0.28²	0.36²/ns	0.26²/ns

M, male; F, female; DM, diabetes mellitus; pTDP43, phosphorylated transactive DNA binding protein 43 (extent/Josephs stage [22]); HPτ, extent/Braak stage [9]; Aβ, extent/Thal phase [10]; αS, extent/Braak stage [26]; CeVL, extent Table 1; ArT, extent Table 1; NpS, extent Table 1; CorP, extent Table 1.

The level of ADNC ranged from low to high. Thirty-four out of the 119 subjects (29%) displayed HPτ pathology with AD distribution but lacked concomitant Aβ pathology thus, fulfilling the criteria for PART. None of these subjects displayed CI (21 females and 13 males). In 28 (24%) cognitively unimpaired subjects, a low level of ADNC was observed (17 males and 11 females). Forty-six (39%) subjects fulfilled the criteria for an intermediate level of ADNC, where the gender was evenly distributed (24 females and 22 males), 19 (41%) of these subjects were demented. Ten subjects (8%) all with CI displayed high level of ADNC (5 females and 5 males), and all of them had DM. One male subject without DM or CI displayed only Aβ; HPτ was not observed in the brain.

None of the 29 subjects with CI displayed only ADNC, and only 6 out of 90 subjects without CI displayed only PART or ADNC. The most common concomitant pathology with ADNC or PART was pTDP43 (seen in 74 subjects), followed by CeVL (seen in 67 subjects). The extent of pTDP43 was significantly lower (MWU test 0.001) in subjects with PART when compared with subjects with ADNC (m±SE; 0.79±0.2 versus 1.65±0.2). The extent of CeVL was significantly higher (MWU test 0.05) in subjects with PART when compared with subjects with ADNC (m±SE; 1.38±0.2 versus 0.88±0.1). One subject without CI displayed five different pathologies (PART + ARTAG +αS + CAA + CeVL), and two subjects with CI displayed six different pathologies (ADNC + ARTAG + pTDP43 +αS + CAA + CeVL).

Some of the alterations seen in combination with the intermediate ADNC were more common in subjects with CI when compared with subjects without CI (pTDP43, in 89 respective 78%; ARTAG, in 68 respective 44%; microscopic infarcts in 21 respective 15%).

In four out of the 19 subjects with intermediate ADNC and clinical signs of CI (14% of all with CI), the extent of concomitant αS pathology was significant and thus considered as causative regarding CI (Braak stage≥4). In five subjects (17% of all with CI), the extent of pTDP43 pathology was considered as causative regarding CI (Josephs stage≥3) fulfilling the criteria for LATE [24]. In as many as six subjects with CI (21% of all with CI) with intermediate ADNC, the extent of CeVL was sufficient to be considered as causative regarding CI; thus, these subjects fulfilled the criteria for VCI.

Further, some concomitant alterations were more common in subjects lacking CI when compared to subjects with CI (αS, in 41 respective 21%, gross brain infarcts in 26 respective 16%, infarcts in deep gray matter in 19 respective 11%). The observation of CeVL did not vary significantly between subjects with or without CI (53/54%). In 42% of subjects with CI, three or more concomitant pathologies were observed in addition to intermediate ADNC compared to 37% of subjects without CI.

DISCUSSION

In the final state, PM, based on our results while studying the brains, vessels, hearts, and kidneys of 119 elderly subjects, the CaVD did not display a causative association with the hallmark lesions of AD. Thus, we were unable to confirm the suggestions that CaVD alters the extent of misfolded proteins, i.e., Aβ and HPτ, our results are in contradiction with frequently encountered statements in line with “ ... growing body of evidence demonstrates an association between vascular risk factors and AD ... ” [1 –6]. There are several explanations to this differing outcome. Several of the studies suggesting an association between AD and CaVD were carried out within the field of epidemiology, having one weakness, the reliability of the clinical diagnosis, both regarding the cause of CI as well as regarding the severity of CaVD. Already in 2009, it was reported that the absolute clinicopathological concordance regarding CI was in Southern Sweden on the level of 49%, suggesting that the cause of CI is not readily assessed clinically applying current diagnostic strategies [46]. Further, regarding the suggestions related to the association between ADNC and CaVD, most reports prior to 2010 are not as such comparable with ours due to significant differences in used methodologies and assessment strategies. The currently recommended, and by us used, method of immunohistochemistry visualizes the misfolded proteins HPτ and Aβ, the constituents of neurofibrillary tangles and neuritic plaques robustly and more efficiently when compared with histochemical stains. Furthermore, we applied here the currently recommended strategy assigning the severity based on regional distribution of misfolded proteins rather than assessing the extent of alterations in a certain brain region [11, 12]. These methodological aspects are highly relevant. The former has facilitated the detection of misfolded proteins in the brain even when they are seen sparsely, whereas the latter has led to a reproducible and reliable assessment of severity of the pathological alterations that are considered to be being causative for CI [9 , 20]. Some of the studies suggesting that there is an association between CaVD and AD are based on radiological methods; thus, they are not as such comparable with what is reported here [1]. Another important difference between our study and previous studies is the age of the study cohort. In this study, we only included subjects within a relatively narrow age span of 10 years, as all alterations of interest, misfolded proteins, and CaVD have a common risk factor of age; thus, the severity of these alterations is expected to increase also with age. Furthermore, our subjects were picked out based on their age from an unselected autopsy cohort where the autopsy was carried out in order to assign the cause of death. Thus, the subjects were not members of any prospective clinical studies. The final difference is the mode of assessing CaVD. It is cumbersome to assess CaVD as a risk factor in vivo. In the industrial world, most patients are under various recommended treatments due to their chronic maladies (hypertension, cardiac dysfunction, dyslipidemia, DM) and follow their treatments in varying degrees. Moreover, the maladies have been diagnosed at various ages, i.e., duration of the disease varies and some maladies have not even been recognized in vivo as has been shown for DM [40, 41]. Based on the above it is difficult to obtain trustworthy results regarding eventual impact of these chronic diseases on the tissues of interest, thus instead of the clinical diagnosis, we looked for tissue alterations caused by CaVD in the brain (CeVL) and in the peripheral organs (ArT, CorP, NpS).

Here, while assessing the vessels, hearts, and kidney, we observed that pathological alterations in these organs were indeed common in this age group. Vessel alterations (ArT) were common and relatively severe (m±SE 4.66±0.09) in our aged cohort and not influenced by gender but noteworthy, significantly influenced by DM (in DM, m±SE 5.22±0.88). It is well known that ArT is associated with hyperlipidemia albeit not assessed by us [47]. Thus, regarding severity of ArT, there are two defined systemic diseases DM and dyslipidemia that are assessable for pharmaceutical intervention. The severity of CeVL and CorP correlated significantly with ArT, and both CeVL and NpS were more severe in males when compared to females. Contrary to the above, quite surprisingly the extent of CeVL, ArT, NpS, and CorP did not differ between subjects with or without CI. Moreover, even in these aged subjects, the extent of CeVL was as such not associated with CI. None of the 29 subjects with CI displayed only CeVL; thus, in this aged cohort, none of the subjects with CI fulfilled the criteria for the entity VaD.

Out of our 119 aged subjects, 29 (24%) displayed CI, but only 10 (34%) subjects displayed a high level of ADNC, demonstrating that they suffered from AD based on current neuropathological consensus criteria.

In as many as 46 (39%) subjects out of the 119, an intermediate level of ADNC was observed and out of these, 19 subjects (41%) displayed CI. One difference, even if not obvious, between those with (19 subjects) and those without (27 subjects) CI with intermediate level of ADNC, was concomitant pathologies seen in the brain. It is well known that ADNC is frequently associated with other, concomitant, pathologies in the elderly, and it has been suggested that concomitant pathology in a setting with intermediate level of ADNC is the cause of observed CI [17 –20]. Here, we were able to confirm this suggestion. Alterations such as pTDP43, ARTAG, and microscopic infarcts were more often seen in subjects with CI, and the number of subjects with more than three concomitant alterations was higher in the subjects with CI. When comparing subjects with and without CI and with intermediate level of ADNC, both ARTAG (68 versus 44%) and pTDP43 (89 versus 78%) were seen more often in subjects with CI when compared with subjects lacking CI. Intermediate ADNC with CeVL, i.e., VCI was diagnosed 21% of all with CI. None of the subjects within this age range with CI displayed “pure” VaD.

LATE, i.e., ADNC with pTDP43, was diagnosed in 17% of all with CI [24]. Moreover, none of the subjects within this age range had a “pure” LATE. Interestingly, a strong positive correlation was observed between pTDP43, HPτ, and Aβ and these associations were not influenced by gender. Noteworthy, the correlation between pTDP43 and HPτ or Aβ was lacking in subjects with DM when compared to subjects without DM. This outcome can certainly be caused by the sparse number of subjects with DM (n = 18), but it has previously been reported that the extent of pTDP43 in the brain correlates with the extent of islet amylin in the pancreas in subjects with DM [48]. Islet amylin is associated with DM, and it has been reported that this misfolded protein is seen in 77% of subjects with DM when compared with 7% of subjects lacking DM [49]. The association between severity of DM and islet amylin is somewhat controversial; specifically, the extent of antibodies directed to islet amylin has not been shown to correlate with gender, age, or duration of the DM [50]. Nevertheless, the association between pTDP43 and DM is still intriguing, particularly as 21% of subjects with intermediate ADNC and CI in our cohort displayed DM when compared with only 7% of subjects with intermediate ADNC but lacking CI. Noteworthy, in the original cohort within this age range, there were five subjects with pure primary pTDP43 alteration, i.e., FTLD cases that we excluded from our study cohort. In summary, both pTDP43 and αS that have previously been reported to be frequently seen in cases with ADNC are also seen in our elderly cohort [17 , 21–24].

Intermediate ADNC with αS pathology, i.e., Lewy body dementia (LBD), was observed in 14% of all with CI, whereas none of the subjects within this age range had a “pure” LBD. In summary, all 29 aged subjects with CI displayed a high or an intermediate level of ADNC. Thirty-four percent suffered from AD (high level of ADNC), 21% from VCI (+intermediate level of ADNC), 17% from LATE (+intermediate level of ADNC), and 14% from LBD (+intermediate level of ADNC). In the remaining four subjects with CI (14% of all with CI), the CI was assigned as being caused by an intermediate level of ADNC and a combination of various alterations listed above. Thus, our results are in line with what was reported from the Harvard Aging Brain Study, i.e., both Aβ and vascular risk (cerebral vascular lesions) are associated with cognitive decline [7, 8].

Intermediate level of ADNC was seen in as many as 46 (39%) subjects, and 27 of these subjects did not display signs of CI. This patient group is certainly worrisome, as due to the increase of HPτ with age as seen here and due to the frequently observed concomitant pathology, they are all at high risk of developing CI.

Generally, misfolded proteins in the brain were common and HPτ, in particular, was seen in all except one subject. An 82-year-old cognitively unimpaired male who displayed some Aβ aggregates in the neocortex (Thal phase 1), some CAA, and some CeVL.

As many as 29% of the whole cohort fulfilled the neuropathological criteria for PART, an entity that is controversial, i.e., merely a stage before ADNC or not [36 , 52]. The mean age at death did not differ between PART and ADNC (m±SE 83.6±0.5 versus 84.4±0.3), and DM was quite evenly distributed (18% versus 14%). The extent of concomitant pTDP43 was lower in subjects with PART when compared with subjects with ADNC, an observation in line with what has been previously reported [52]. A significant difference was observed regarding gender, namely, 48% of the subjects with ADNC were males when compared with 38% of subjects with PART; thus, PART seems to be more common in females. Noteworthy, none of our subjects with PART displayed CI in this elderly cohort. The extent of HPτ pathology seen in PART (Braak stage≤IV) is not detectable by imaging and there are no reliable biomarkers; thus, the subjects with PART and lacking CI are “diagnosed” only by a PM neuropathological investigation. Thus, the identification of a subject with PART is significantly influenced by the rate of neuropathological investigations carried out on the cognitively unimpaired deceased. In the current setting, when autopsies are seldom carried out and the neuropathological investigations are carried out only when neurological alterations are registered during life, the entity PART is close to non-existent. Our results, however, do indicate that the entity PART does exist and seems to be more common in females. Noteworthy, when PART evolves (Braak stage IV) and CI is noted, the diagnosis is what was earlier known as the tangle predominant (only) dementia [53]. With the age-related increase in the extent of HPτ as seen here, it is not excluded that the relatively uncommon diagnosis of PART displaying CI, i.e., tangle predominant dementia, will be more common.

In conclusion, while assessing 119 subjects within the age range of 80 to 89 years at death, all displayed pathological alterations in the periphery and in the brain. Regarding the peripheral organs, the vessels showed significant pathology. This was in line with the final cause of death being assigned as cardiovascular in 64% of the subjects. In all subjects, misfolded proteins were observed in the brain and the majority displayed not one but several alterations. Twenty-nine subjects (24% of the study cohort) displayed CI during life. Out of these, 34% (10 subjects) were assigned suffering from definite AD, displaying a high level of ADNC. Out of the remaining subjects with CI, 21% suffered from VCI (CeVL + intermediate ADNC), 17% from LATE (pTDP43 + intermediate ADNC), and 14% from LBD (αS + intermediate ADNC). Noteworthy, a substantial number of cognitively unimpaired subjects displayed intermediate ADNC (23%) and should certainly be considered as being at risk of developing CI. Some of these subjects with intermediate ADNC could under certain circumstances develop LATE (+pTDP43), VCI (+CeVL), or AD as the extent of HPτ increases with age, even within this elderly population. Twenty-nine percent of our cohort fulfilled the neuropathological criteria for PART and also this patient group might increase due to an increase in the HPτ pathology. Based on our results, two brain alterations, pTDP43 and CeVL, as opposed to ADNC and PART, were influenced by DM. Moreover, CeVL correlated significantly with ArT, which is associated with dyslipidemia [47]. Thus, the suggestion that systemic diseases such as DM and CaVD are risk factors for CI is certainly in line with our observations [54]. The distinction between AD and CI with mixed pathology, i.e., ADNC + CeVL + pTDP43 + other alterations, is of importance and needs to be acknowledged. Noteworthy, the entity “mixed dementia” was already described 20 years ago and still it is not fully acknowledged [55]. The extent of ADNC, as shown by us, is not influenced by chronic diseases such as CaVD, DM, unlike concomitant alterations such as CeVL and eventual pTDP43. Thus, with proper treatment of systemic chronic diseases such as DM, dyslipidemia, and CaVD, the current 24% of subjects with CI in the age group of 80 to 89 years could be limited to 19% (preventing development of CeVL), and eventually even more. Contrary to the above, a poor intervention might lead to a worrisome increase, i.e., the incidence of CI within this age group might be as high as 47% (all subjects with high and intermediate level of ADNC).

Footnotes

ACKNOWLEDGMENTS

The subjects included and/or their relatives had given their consent for the use of the tissue, and the study was authorized by the regional Ethics Committee of Uppsala, Sweden # 2011, 2015/286. We thank Statisticon for assisting in the statistical analysis and Meena Strömqvist for her critical reading of the manuscript. This study was funded by local grants from Uppsala University Hospital and by Hans Gabriel and Alice Trolle-Wachtmeister Foundation.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

References

Newman

, Fitzpatrick

, Lopez

, Jackson

, Lyketsos

, Jagust

, Ives

, Dekosky

, Kuller

(2005) Dementia and Alzheimer’s disease incidence in relationship to cardiovascular disease in the Cardiovascular Health Study cohort, J Am Geriatr Soc 53, 1101–1107.

Stampfer

(2006) Cardiovascular disease and Alzheimer’s disease: Common links, J Intern Med 260, 211–223.

Guerchet

, Aboyans

, Nubukpo

, Lacroix

, Clément

, Preux

(2011) Ankle-brachial index as a marker of cognitive impairment and dementia in general population. A systematic review, Atherosclerosis 216, 251–257.

Wolters

, Segufa

, Darweesh

SKL

, Bos

, Ikram

, Sabayan

, Hofman

, Sedaghat

(2018) Coronary heart disease, heart failure, and the risk of dementia: A systematic review and meta-analysis, Alzheimers Dement 14, 1493–1504.

Honig

, Kukull

, Mayeux

(2005) Atherosclerosis and AD: Analysis of data from the US National Alzheimer’s Coordinating Center, Neurology 64, 494–500.

Yarchoan

, Xie

, Kling

, Toledo

, Wolk

, Lee

, Van Deerlin

, Lee

VMY

, Trojanowski

, Arnold

(2012) Cerebrovascular atherosclerosis correlates with Alzheimer pathology in neurodegenerative dementias, Brain 135, 3749–3756.

Rabin

, Schultz

, Hedden

, Viswanathan

, Marshall

, Kilpatrick

, Klein

, Buckley

, Yang

, Properzi

, Rao

, Kirn

, Papp

, Rentz

, Johnson

, Sperling

, Chhatwal

(2018) Interactive associations of vascular risk and β-Amyloid burden with cognitive decline in clinically normal elderly individuals: Findings from the harvard aging brain study, JAMA Neurol 75, 1124–1131.

Rabin

, Yang

, Schultz

, Hanseeuw

, Hedden

, Viswanathan

, Gatchel

, Marshall

, Kilpatrick

, Klein

, Rao

, Buckley

, Yau

WYW

, Kirn

, Rentz

, Johnson

, Sperling

, Chhatwal

(2019) Vascular risk and β-amyloid are synergistically associated with cortical tau, Ann Neurol 85, 272–279.

Braak

, Alafuzoff

, Arzberger

, Kretzschmar

, Del Tredici

(2006) Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry, Acta Neuropathol 112, 389–404.

10.

Thal

, Rüb

, Orantes

, Braak

(2002) Phases of A beta-deposition in the human brain and its relevance for the development of AD, Neurology 58, 1791–800.

11.

Montine

, Phelps

, Beach

, Bigio

, Cairns

, Dickson

, Duyckaerts

, Frosch

, Masliah

, Mirra

, Nelson

, Schneider

, Thal

, Trojanowski

, Vinters

, Hyman

; National Institute on Aging; Alzheimer’s Association (2012) National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: A practical approach, Acta Neuropathol 123, 1–11.

12.

Hyman

, Phelps

, Beach

, Bigio

, Cairns

, Carrillo

, Dickson

, Duyckaerts

, Frosch

, Masliah

, Mirra

, Nelson

, Schneider

, Thal

, Thies

, Trojanowski

, Vinters

, Montine

(2012) National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease, Alzheimers Dement 8, 1–13.

13.

Braak

, Thal

, Ghebremedhin

, Del Tredici

(2011) Stages of the pathologic process in Alzheimer disease: Age categories from 1 to 100 years, J Neuropathol Exp Neurol 70, 960–969.

14.

Elobeid

, Libard

, Leino

, Popova

, Alafuzoff

(2016) Altered proteins in the aging brain, J Neuropathol Exp Neurol 75, 316–325.

15.

Alafuzoff

, Arzberger

, Al-Sarraj

, Bodi

, Bogdanovic

, Braak

, Bugiani

, Del-Tredici

, Ferrer

, Gelpi

, Giaccone

, Graeber

, Ince

, Kamphorst

, King

, Korkolopoulou

, Kovács

, Larionov

, Meyronet

, Monoranu

, Parchi

, Patsouris

, Roggendorf

, Seilhean

, Tagliavini

, Stadelmann

, Streichenberger

, Thal

, Wharton

, Kretzschmar

(2008) Staging of neurofibrillary pathology in Alzheimer’s disease: A study of the BrainNet Europe Consortium, Brain Pathol 18, 484–496.

16.

Alafuzoff

, Thal

, Arzberger

, Bogdanovic

, Al-Sarraj

, Bodi

, Boluda

, Bugiani

, Duyckaerts

, Gelpi

, Gentleman

, Giaccone

, Graeber

, Hortobagyi

, Höftberger

, Ince

, Ironside

, Kavantzas

, King

, Korkolopoulou

, Kovács

, Meyronet

, Monoranu

, Nilsson

, Parchi

, Patsouris

, Pikkarainen

, Revesz

, Rozemuller

, Seilhean

, Schulz-Schaeffer

, Streichenberger

, Wharton

, Kretzschmar

(2009) Assessment of beta-amyloid deposits in human brain: A study of the BrainNet Euroe Consortium. Acta Neuropathol 117, 309–320.

17.

Kovacs

, Alafuzoff

, Al-Sarraj

, Arzberger

, Bogdanovic

, Capellari

, Ferrer

, Gelpi

, Kövari

, Kretzschmar

, Nagy

, Parchi

, Seilhean

, Soininen

, Troakes

, Budka

(2008) Mixed brain pathologies in dementia: The BrainNet Europe consortium experience, Dement Geriatr Cogn Disord 26, 343–350.

18.

Snowdon

, Greiner

, Mortimer

, Riley

, Greiner

, Markesbery

(1997) Brain infarction and the clinical expression of Alzheimer disease. The Nun Study, JAMA 277, 813–817.

19.

Launer

, Petrovitch

, Ross

, Markesbery

, White

(2008) AD brain pathology: Vascular origins? Results from the HAAS autopsy study, Neurobiol Aging 29, 1587–1590.

20.

, Jagust

(2012) Vascular burden and Alzheimer disease pathologic progression. Alzheimer’s Disease Neuroimaging Initiative, Neurology 79, 1349–1355.

21.

Uchikado

, Lin

, DeLucia

, Dickson

(2006) Alzheimer disease with amygdala Lewy bodies: A distinct form of alpha-synucleinopathy, J Neuropathol Exp Neurol 65, 685–697.

22.

Josephs

, Murray

, Whitwell

, Parisi

, Petrucelli

, Jack

, Petersen

, Dickson

(2014) Staging TDP-43 pathology in Alzheimer’s disease, Acta Neuropathol 127, 441–450.

23.

Josephs

, Murray

, Whitwell

, Tosakulwong

, Weigand

, Petrucelli

, Liesinger

, Petersen

, Parisi

, Dickson

(2016) Updated TDP-43 in Alzheimer’s disease staging scheme, Acta Neuropathol 131, 571–585.

24.

Nelson

, Dickson

, Trojanowski

, Jack

, Boyle

, Arfanakis

, Rademakers

, Alafuzoff

, Attems

, Brayne

, Coyle-Gilchrist

ITS

, Chui

, Fardo

, Flanagan

, Halliday

, Hokkanen

SRK

, Hunter

, Jicha

, Katsumata

, Kawas

, Keene

, Kovacs

, Kukull

, Levey

, Makkinejad

, Montine

, Murayama

, Murray

, Nag

, Rissman

, Seeley

, Sperling

, White Iii

, Yu

, Schneider

(2019) Limbic-predominant age-related TDP-43 encephalopathy (LATE): Consensus working group report, Brain 142, 1503–1527.

25.

McKeith

, Galasko

, Kosaka

, Perry

, Dickson

, Hansen

, Salmon

, Lowe

, Mirra

, Byrne

, Lennox

, Quinn

, Edwardson

, Ince

, Bergeron

, Burns

, Miller

, Lovestone

, Collerton

, Jansen

, Ballard

, de Vos

, Wilcock

, Jellinger

, Perry

(1996) Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): Report of the consortium on DLB international workshop, Neurology 47, 1113–1124.

26.

Alafuzoff

, Ince

, Arzberger

, Al-Sarraj

, Bell

, Bodi

, Bogdanovic

, Bugiani

, Ferrer

, Gelpi

, Gentleman

, Giaccone

, Ironside

, Kavantzas

, King

, Korkolopoulou

, Kovács

, Meyronet

, Monoranu

, Parchi

, Parkkinen

, Patsouris

, Roggendorf

, Rozemuller

, Stadelmann-Nessler

, Streichenberger

, Thal

, Kretzschmar

(2009) Staging/typing of Lewy body related alpha-synuclein pathology: A study of the BrainNet Europe Consortium, Acta Neuropathol 117, 635–652.

27.

Alafuzoff

, Pikkarainen

, Neumann

, Arzberger

, Al-Sarraj

, Bodi

, Neumann

, Arzberger

, Al-Sarraj

, Bodi

, Bogdanovic

, Bugiani

, Ferrer

, Gelpi

, Gentleman

, Giaccone

, Graeber

, Hortobagyi

, Ince

, Ironside

, Kavantzas

, King

, Korkolopoulou

, Kovács

, Meyronet

, Monoranu

, Nilsson

, Parchi

, Patsouris

, Revesz

, Roggendorf

, Rozemuller

, Seilhean

, Streichenberger

, Thal

, Wharton

, Kretzschmar

(2015) Neuropathological assessments of the pathology in frontotemporal lobar degeneration with TDP43-positive inclusions: An inter-laboratory study by the BrainNet Europe consortium, J Neural Transm (Vienna) 122, 957–972.

28.

Gorelick

, Scuteri

, Black

, Decarli

, Greenberg

, Iadecola

, Launer

, Laurent

, Lopez

, Nyenhuis

, Petersen

, Schneider

, Tzourio

, Arnett

, Bennett

, Chui

, Higashida

, Lindquist

, Nilsson

, Roman

, Sellke

, Seshadri

; American Heart Association Stroke Council, Council on Epidemiology and Prevention, Council on Cardiovascular Nursing, Council on Cardiovascular Radiology and Intervention, and Council on Cardiovascular Surgery and Anesthesia (2011) Vascular contributions to cognitive impairment and dementia: A statement for healthcare professionals from The American Heart Association/American Stroke Association, Stroke 42, 2672–2713.

29.

Kalaria

, Maestre

, Arizaga

, Friedland

, Galasko

, Hall

, Luchsinger

, Ogunniyi

, Perry

, Potocnik

, Prince

, Stewart

, Wimo

, Zhang

, Antuono

; World Federation of Neurology Dementia Research Group (2008) Alzheimer’s disease and vascular dementia in developing countries: Prevalence, management, and risk factors, Lancet Neurol 7, 812–826.

30.

van der Flier

, Skoog

, Schneider

, Pantoni

, Mok

, Chen

CLH

, Scheltens

(2018) Vascular cognitive impairment, Nat Rev Dis Primers 15, 4.

31.

Sweeney

, Montagne

, Sagare

, Nation

, Schneider

, Chui

, Harrington

, Pa

, Law

, Wang

DJJ

, Jacobs

, Doubal

, Ramirez

, Black

, Nedergaard

, Benveniste

, Dichgans

, Iadecola

, Love

, Bath

, Markus

, Salman

, Allan

, Quinn

, Kalaria

, Werring

, Carare

, Touyz

, Williams

SCR

, Moskowitz

, Katusic

, Lutz

, Lazarov

, Minshall

, Rehman

, Davis

, Wellington

, González

, Yuan

, Lockhart

, Hughes

, Chen

CLH

, Sachdev

, O’Brien

, Skoog

, Pantoni

, Gustafson

, Biessels

, Wallin

, Smith

, Mok

, Wong

, Passmore

, Barkof

, Muller

, Breteler

MMB

, Román

, Hamel

, Seshadri

, Gottesman

, van Buchem

, Arvanitakis

, Schneider

, Drewes

, Hachinski

, Finch

, Toga

, Wardlaw

, Zlokovic

(2019) Vascular dysfunction-The disregarded partner of Alzheimer’s disease, Alzheimers Dement 15, 158–167.

32.

Alafuzoff

, Gelpi

, Al-Sarraj

, Arzberger

, Attems

, Bodi

, Bogdanovic

, Budka

, Bugiani

, Englund

, Ferrer

, Gentleman

, Giaccone

, Graeber

, Hortobagyi

, Höftberger

, Ironside

, Jellinger

, Kavantzas

, King

, Korkolopoulou

, Kovács

, Meyronet

, Monoranu

, Parchi

, Patsouris

, Roggendorf

, Rozemuller

, Seilhean

, Streichenberger

, Thal

, Wharton

, Kretzschmar

(2012) The need to unify neuropathological assessments of vascular alterations in the ageing brain: Multicentre survey by the BrainNet Euroe consortium. Exp Gerontol 47, 825–833.

33.

Esiri

, Wilcock

, Morris

(1997) Neuropathological assessment of the lesions of significance in vascular dementia, J Neurol Neurosurg Psychiatry 63, 749–753.

34.

Esiri

(2000) Which vascular lesions are of importance in vascular dementia? Ann N Y Acad Sci 903, 239–243.

35.

Kovacs

, Ferrer

, Grinberg

, Alafuzoff

, Attems

, Budka

, Cairns

, Crary

, Duyckaerts

, Ghetti

, Halliday

, Ironside

, Love

, Mackenzie

, Munoz

, Murray

, Nelson

, Takahashi

, Trojanowski

, Ansorge

, Arzberger

, Baborie

, Beach

, Bieniek

, Bigio

, Bodi

, Dugger

, Feany

, Gelpi

, Gentleman

, Giaccone

, Hatanpaa

, Heale

, Hof

, Hofer

, Hortobágyi

, Jellinger

, Jicha

, Ince

, Kofler

, Kövari

, Kril

, Mann

, Matej

, McKee

, McLean

, Milenkovic

, Montine

, Murayama

, Lee

, Rahimi

, Rodriguez

, Rozemüller

, Schneider

, Schultz

, Seeley

, Seilhean

, Smith

, Tagliavini

, Takao

, Thal

, Toledo

, Tolnay

, Troncoso

, Vinters

, Weis

, Wharton

, White

3rd , Wisniewski

, Woulfe

, Yamada

, Dickson

(2016) Aging-related tau astrogliopathy (ARTAG): Harmonized evaluation strategy, Acta Neuropathol 131, 87–102.

36.

Crary

, Trojanowski

, Schneider

, Abisambra

, Abner

, Alafuzoff

, Arnold

, Attems

, Beach

, Bigio

, Cairns

, Dickson

, Gearing

, Grinberg

, Hof

, Hyman

, Jellinger

, Jicha

, Kovacs

, Knopman

, Kofler

, Kukull

, Mackenzie

, Masliah

, McKee

, Montine

, Murray

, Neltner

, Santa-Maria

, Seeley

, Serrano-Pozo

, Shelanski

, Stein

, Takao

, Thal

, Toledo

, Troncoso

, Vonsattel

, White

3rd , Wisniewski

, Woltjer

, Yamada

, Nelson

(2014) Primary age-related tauopathy (PART): A common pathology associated with human aging, Acta Neuropathol 128, 755–766.

37.

Pal

, Mukadam

, Petersen

, Cooper

(2018) Mild cognitive impairment and progression to dementia in people with diabetes, prediabetes and metabolic syndrome: A systematic review and meta-analysis, Soc Psychiatry Psychiatr Epidemiol 53, 1149–1160.

38.

Wijesinghe

, Shankar

, Yasha

, Gorrie

, Amaratunga

, Hulathduwa

, Kumara

, Samarasinghe

, Suh

, Steinbusch

, De

Silva KR

(2016) Vascular contributions in Alzheimer’s disease-related neuropathological changes: First autopsy evidence from a South Asian aging population, J Alzheimers Dis 54, 1607–1618.

39.

Ford

, Greenslade

, Paudyal

, Bremner

, Smith

, Banerjee

, Sadhwani

, Rooney

, Oliver

, Cassell

(2018) Predicting dementia from primary care records: A systematic review and meta-analysis, PLoS One 13, e0194735.

40.

Dall

, Yang

, Halder

, Franz

, Byrne

, Semilla

, Chakrabarti

, Stuart

(2016) Type 2 diabetes detection and management among insured adults, Popul Health Metr 21, 14–43.

41.

Eades

, France

, Evans

(2016) Prevalence of impaired glucose regulation in Europe: A meta-analysis, Eur J Public Health 26, 699–706.

42.

Qiu

(2012) Preventing Alzheimer’s disease by targeting vascular risk factors: Hope and gap, J Alzheimers Dis 32, 721–731.

43.

Kimmelstiel

, Wilson

(1936) Benign and malignant hypertension and nephrosclerosis: A clinical and pathological study, Am J Pathol 12, 45–82.

44.

Lowe

, Bate

(1948) The diameter of cardiac muscle fibres; a study of the diameter of muscle fibres in the left ventricular enlargement of simple hypertension, Med J Aust 1, 467–469.

45.

Sethi

, D’Agati

, Nast

, Fogo

, De Vriese

, Markowitz

, Glassock

, Fervenza

, Seshan

, Rule

, Racusen

, Radhakrishnan

, Winearls

, Appel

, Bajema

, Chang

, Colvin

, Cook

, Hariharan

, Herrera Hernandez

, Kambham

, Mengel

, Nath

, Rennke

, Ronco

, Rovin

, Haas

(2017) A proposal for standardized grading of chronic changes in native kidney biopsy specimens, Kidney Int 91, 787–789.

46.

Brunnström

, Englund

(2009) Clinicopathological concordance in dementia diagnostics, Am J Geriatr Psychiatry 17, 664–670.

47.

McNamara

, Howell

(1992) Epidemiologic data linking diet to hyperlipidemia and arteriosclerosis, Semin Liver Dis 12, 347–355.

48.

Leino

, Popova

, Alafuzoff

(2017) Transactive DNA binding protein 43 rather than other misfolded proteins in the brain is associated with islet amyloid polypeptide in pancreas in aged subjects with diabetes mellitus, J Alzheimers Dis 59, 43–56.

49.

Clark

, Saad

, Nezzer

, Uren

, Knowler

, Bennett

, Turner

(1990) Islet amyloid polypeptide in diabetic and non-diabetic Pima Indians, Diabetologia 33, 285–289.

50.

Clark

, Yon

, de Koning

, Holman

(1991) Autoantibodies to islet amyloid polypeptide in diabetes, Diabet Med 8, 668–673.

51.

Duyckaerts

, Braak

, Brion

, Buée

, Del Tredici

, Goedert

, Halliday

, Neumann

, Spillantini

, Tolnay

, Uchihara

(2015) PART is part of Alzheimer disease, Acta Neuropathol 129, 749–756.

52.

Josephs

, Murray

, Tosakulwong

, Whitwell

, Knopman

, Machulda

, Weigand

, Boeve

, Kantarci

, Petrucelli

, Lowe

, Jack

Jr , Petersen

, Parisi

, Dickson

(2017) Tau aggregation influences cognition and hippocampal atrophy in the absence of beta-amyloid: A clinico-imaging-pathological study of primary age-related tauopathy (PART), Acta Neuropathol 133, 705–715.

53.

Jellinger

, Bancher

(1998) Senile dementia with tangles (tangle predominant form of senile dementia), Brain Pathol 8, 367–376.

54.

, Zhao

, Liu

, Schiman

, Lloyd-Jones

, Daviglus

, Liu

, Garside

, Stamler

, Fries

, Shih

, Allen

(2019) Favorable cardiovascular health at young and middle ages and dementia in older age-the CHA Study, J Am Heart Assoc 8, e009730.

55.

Korczyn

(2002) Mixed dementia–the most common cause of dementia, Ann N Y Acad Sci 977, 129–134.