Abstract

PICALM, the gene encoding phosphatidylinositol-binding clathrin assembly (picalm) protein, was recently shown to be associated with risk of Alzheimer disease (AD). Picalm is a key component of clathrin-mediated endocytosis. It recruits clathrin and adaptor protein 2 (AP-2) to the plasma membrane and, along with, AP-2 recognizes target proteins. The attached clathrin triskelions cause membrane deformation around the target proteins enclosing them within clathrin-coated vesicles to be processed in lysosomes or endosomes. We examined the distribution of picalm in control and AD brain tissue and measured levels of picalm messenger RNA (mRNA) by real-time polymerase chain reaction. Immunolabeling of brain tissue showed that picalm is predominately present in endothelial cells. This was further supported by the demonstration of picalm in human cerebral microvascular cells grown in culture. Picalm mRNA was elevated in relation to glyceraldehyde-3-phosphate dehydrogenase but not factor VIII-related antigen or CD31 mRNA in the frontal cortex in AD. No change was seen in the temporal cortex or thalamus. The transport of Aβ across vessel walls and into the bloodstream is a major pathway of Aβ removal from the brain and picalm is ideally situated within endothelial cells to participate in this process. Further research is needed to determine whether PICALM expression is influenced by Aβ levels and whether it affects Aβ uptake and transport by endothelial cells.

Introduction

Two recent genome-wide association studies of Alzheimer disease (AD) demonstrated an association with PICALM variants (1, 2). PICALM was originally identified as the clathrin assembly lymphoid myeloid leukemia (CALM) gene in studies of a rare translocation present in lymphoid and myeloid acute leukemias (3). The gene product phosphatidylinositol-binding clathrin assembly (picalm) protein plays a key role in endocytosis (4), which is important in processes such as the regulation of receptors, synaptic transmission and the removal of apoptotic cells (5). Processing of the amyloid precursor protein (APP) after endocytosis suggests involvement of this process in the regulation of β-amyloid (Aβ) levels (6-9). The transport of endocytosed APP into lysosomes is reported to result in APP degradation entry into endosomes and Aβ production (9).

One mechanism of endocytosis is dependent on clathrin-coated vesicles (CCVs), which are involved in receptor-mediated endocytosis at the plasma membrane and in the intracellular movement of macromolecules (10, 11). Clathrin-coated vesicles are formed on the cytoplasmic side of the plasma membrane or at the trans-Golgi network after the attachment of clathrin triskelions. The polyhedral lattices formed by these triskelions lead to the deformation of the membrane and vesicle formation (4, 5). Clathrin-coated vesicles subsequently fuse with either endosomes, in which internalized target proteins are modified or recycled back to the plasma membrane, or with lysosomes, in which case the target protein is degraded (5, 12).

The attachment of clathrin triskelions to the membrane is by means of APs, of which there are 2 types: tetrameric (AP-1 to -4) and monomeric (AP180 and picalm) (13). In addition to binding to clathrin, APs interact with membrane proteins containing specific sequences that mark them as proteins for internalization (14, 15). There are 3 forms of picalm (i.e. full-length [652 residues] and 2 shorter forms [632 and 610 residues]), which is an important component of clathrin-mediated endocytosis. Its N-terminal binds to phosphatidylinositol-4,5-biphosphate present in the plasma membrane and its C-terminal to clathrin and AP-2, recruiting both to the membrane (4). Picalm is reported to be present within neurons, astrocytes, and oligodendrocytes (4, 10, 11, 16), and alterations in its levels affect clathrin-mediated endocytosis. Endocytosis can be blocked by picalm overexpression (11) and by degradation of picalm and AP-2 by calpain and caspase proteases, the levels of which are increased in AD (13, 17).

Here, we examined the expression and distribution of picalm in control and AD brains. Our findings suggest that it may contribute to Aβ clearance from the brain parenchyma into the bloodstream.

Materials and Methods

Tissue from control and AD brains was selected from the South West Dementia Brain Bank. The brains had been divided midsagittally at autopsy: the left half had been sliced and frozen at −80°C for biochemical studies and the right half had been fixed in formalin for detailed neuropathological assessment. Diagnosis of AD was made according to Consortium to Establish a Registry for Alzheimer's Disease criteria (18). According to National Institute on Aging-Reagan criteria (19), 17 of these 19 cases had a high likelihood that the dementia was due to AD. The remaining 2 cases (ID numbers 23 and 29; Table 1) had an intermediate likelihood that dementia was due to AD. Aβ load and cerebral amyloid angiopathy (CAA) scores were determined as previously described (20, 21) (Table 1). Cerebral amyloid angiopathy was graded in arteriolar vessels in the cortex and in the leptomeninges according to the method of Olichney et al (21).

TABLE 1.

Profiles of Cases Studied

Western Blot

To determine the specificity of the picalm antibody, brain tissue homogenates were prepared in sodium dodecyl sulfate buffer as previously described (22) and were run alongside glutathione-S-transferase (GST)-tagged recombinant picalm protein (AbNova, Taipei, Taiwan) on a 4% to 20% Tris-HCl precast gel (Bio-Rad, Hertfordshire, UK; 150 V, 1 hour). Protein was transferred to nitrocellulose membrane (Anachem, Luton, UK; 20 V, overnight [ON] at 4°C) and detected using the Millipore SNAP i.d. protein detection system (Millipore, Billerica, MA). The membrane was blocked in 0.5% milk powder in Tris-buffered saline/0.05% Tween-20, incubated with picalm (1:50, 10 minutes; Calm C-18; Santa Cruz Biotechnology, Heidelberg, Germany) and with peroxidase antigoat (1:10,000, 10 minutes; Vector Laboratories, Peterborough, UK). Visualization of the target protein was by enhanced chemiluminescence using the Amersham ECL detection reagents (GE Healthcare, Buckinghamshire, UK).

Immunohistochemistry

Sections 7 μm thick from formalin-fixed, paraffin-embedded blocks of the frontal and temporal lobes from 7 control and 7 AD brains were immunostained for picalm. The sections of the frontal lobe included the superior and middle frontal gyri (Brodmann area 6), cingulate cortex (Brodmann area 24), paracentral lobule (Brodmann area 32), and underlying white matter. Those of temporal lobe included the parahippocampal gyrus (Brodmann area 36), and inferior (Brodmann area 20), middle (Brodmann area 21), and superior temporal (Brodmann area 22) gyri and the hippocampus. Dewaxed and hydrated sections were immersed in methanol containing 3% H2O2 (30 minutes), boiled in sodium citrate buffer (pH 6), blocked in 10% normal rabbit serum (20 minutes), and incubated with antipicalm antibody (1:800, ON; Calm C-18; Santa Cruz Biotechnology). This was followed by incubations with biotinylated antigoat secondary antibody (1:100, 20 minutes; Vector Laboratories), with avidin-biotin horseradish peroxidase complex (20 minutes; VectaElite ABC; Vector Laboratories) and with 3,3′-diaminobenzidine (DAB, 10 minutes; Vector Laboratories). All incubations were conducted at room temperature. Sections were counterstained with hematoxylin, dehydrated, cleared, and mounted.

Double Immunofluorescent Labeling of Brain Tissue

Fluorescein tyramide-labeled factor VIII-related antigen/von Willebrand factor (FVIIIRA/VWF) was combined with streptavidin Alexa Fluor 555 labeling of biotinylated picalm. Sections were initially treated as previously mentioned. After treatment with sodium citrate buffer, sections were incubated in 0.12% potassium permanganate in phosphate-buffered saline (PBS; 25 minutes) and in 1% oxalic acid and 1% potassium pyrosulfate in PBS (15 minutes) to reduce autofluorescence. Sections were washed and labeled for FVIIIRA/VWF (VWF; Abcam, Cambridge, UK) using the tyramide signal amplification (TSA) fluorescence system (Perkin Elmer, Milan, Italy). This involved initial immersion in Tris-sodium chloride buffer containing 0.5% TSA blocking reagent for 30 minutes and subsequent incubation in primary antibody (1:800; ON, room temperature), peroxidase antirabbit antibody (1:200; 30 minutes), and fluorescein-labeled tyramide (1:50; 10 minutes). This was followed by ON incubation with antipicalm (1:100), 20-minute incubation with biotinylated antigoat (1:100) and 1-hour incubation with streptavidin Alexa Fluor 555 (Invitrogen, Paisley, UK). Sections were mounted in Vectashield mounting medium (Vector Laboratories), and images were acquired using a confocal laser scanning microscope (Nikon Instruments, The Netherlands).

The TSA biotin system (Perkin Elmer) was used for double immunofluorescent labeling of picalm and Aβ (4G8; Covance Cambridge Bioscience, Cambridge, UK). After treatment with sodium citrate buffer as previously mentioned, sections were blocked in TSA blocking buffer (30 minutes), incubated with antipicalm (1:800, ON), biotinylated antigoat (30 minutes), streptavidin-horseradish peroxidase (30 minutes), biotinyl tyramide (10 minutes), and streptavidin Alexa Fluor 555 (1 hour). Sections were washed and incubated ON with 4G8 (1:2000) and with donkey antimouse Alexa Fluor 488 (1 hour).

Double Immunofluorescent Labeling of Cerebrovascular Endothelial Cells

Human brain microvascular endothelial cells (TCS Cellworks, Buckingham, UK) were grown on fibronectin-coated coverslips in endothelial cell medium supplemented with 5% fetal bovine serum and 1% growth supplement (TCS Cellworks) at 37°C in 5% CO2. Cells were fixed in 4% paraformaldehyde (10 minutes; Alfa Aesar, Lancashire, UK), blocked and permeabilized in a solution containing 5% normal donkey serum and 0.1% Triton X-100 in PBS (20 minutes), and then incubated ON at 4°C with FVIIIRA/VWF (1:100) and picalm (1:50) antibodies. Cells were subsequently incubated with donkey antirabbit Alexa Fluor 488 (1 hour), with biotinylated antigoat antibody (1:100, 20 minutes), and with streptavidin Alexa Fluor 555 (1 hour). Cells were mounted in Vectashield mounting medium, and images were acquired using a confocal laser scanning microscope.

RNA Extraction and Reverse Transcription

RNA extraction from the frontal and temporal cortex and from the thalamus of 20 controls and 19 AD cases was performed as previously described (23). Profiles of the cases are listed in Table 1. There were no significant differences in postmortem delay (p = 0.832) or age (p = 0.433) between the 2 groups. RNA concentration was determined using a Ribogreen RNA quantification kit (Invitrogen). RNA was reverse transcribed using the High Capacity c-DNA Archive Kit (Applied Biosystems, Foster City, CA), and the concentration of the resulting complementary DNA determined using the Picogreen DNA quantification kit (Invitrogen). These kits were used according to the manufacturers' instructions.

Real-Time Polymerase Chain Reaction

The expression of PICALM and the calibrator genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FVIIIRA/VWF, and CD31/platelet endothelial cell adhesion molecule were measured using the ABI 7000 sequence detection system (Hs00200318_m1, Hs99999905_m1, Hs01109438_m1, and Hs01065279_m1, respectively; Applied Biosystems). The 20-μL reaction mixture comprised SensiMix dT (Quantace, London, UK), TaqMan gene expression assay (Applied Biosystems), and 10 ng of complementary DNA. Each sample was analyzed in triplicate using the following program settings: 50°C (2 minutes), 95°C (10 minutes), and 40 cycles of 95°C (15 seconds) and 60°C (1 minute). PICALM expression in relation to each calibrator gene was calculated (ΔCt) and used to determine the fold change in PICALM expression (2−ΔΔCt) in each case relative to the mean expression in controls.

Statistical Analysis

The Mann-Whitney U test was used to assess the significance of changes in PICALM expression between AD and control groups. p values < 0.05 were regarded as significant.

Results

Western Blot Analysis and Immunolabeling

The specificity of the picalm antibody was confirmed by Western blot. The antibody recognized the GST-tagged recombinant protein (∼93 kd) and picalm in brain tissue homogenates (∼62-72 kd; Fig. 1). The 652- and 632-amino acid forms of picalm have very similar molecular weights (∼69 and ∼71 kd) and could not be resolved by Western blot.

FIGURE 1.

Specificity of the picalm antibody was tested by Western blot. Antipicalm recognizes GST-tagged recombinant picalm (∼93 kd) in lane 1 and picalm in brain tissue homogenates in lanes 2 and 3 (∼62-72 kd). Both full-length picalm (71/72 kd) and the shorter isoform (62-66 kd) are detected. The third isoform (∼69 kd) could not be separated from the 71/72-kd full-length picalm band.

FIGURE 1.

Specificity of the picalm antibody was tested by Western blot. Antipicalm recognizes GST-tagged recombinant picalm (∼93 kd) in lane 1 and picalm in brain tissue homogenates in lanes 2 and 3 (∼62-72 kd). Both full-length picalm (71/72 kd) and the shorter isoform (62-66 kd) are detected. The third isoform (∼69 kd) could not be separated from the 71/72-kd full-length picalm band.

Immunolabeling of human brain tissue showed that picalm was largely restricted to blood vessel walls (Fig. 2). Picalm antibody labeled vessels throughout the gray and white matter and leptomeninges. There was only weak neuronal or glial labeling (Fig. 2A), and there was no evidence of picalm in Aβ plaques or neurofibrillary tangles.

FIGURE 2.

Sections of human brain tissue immunolabeled with antipicalm. (A) Picalm is present in blood vessel walls in a section of normal frontal cortex. Neurons are only very weakly labeled (arrows). (B, C) The predominant localization of picalm in the walls of blood vessels is also evident in normal temporal cortex (B) and Alzheimer disease temporal cortex (C). Magnifications: ×50.

FIGURE 2.

Sections of human brain tissue immunolabeled with antipicalm. (A) Picalm is present in blood vessel walls in a section of normal frontal cortex. Neurons are only very weakly labeled (arrows). (B, C) The predominant localization of picalm in the walls of blood vessels is also evident in normal temporal cortex (B) and Alzheimer disease temporal cortex (C). Magnifications: ×50.

The location of picalm within the vessel walls was shown by double immunofluorescent labeling to be associated with endothelial cells: the distribution of picalm closely followed that of the endothelial cell marker FVIIIRA/VWF (Figs. 3A-F). The expression of picalm by cerebrovascular endothelial cells was confirmed by immunofluorescent labeling of human brain microvascular endothelial cells grown in culture (Figs. 3G-I). Double immunofluorescent labeling of picalm and Aβ confirmed the absence of picalm from plaques and showed good preservation of picalm even in vessels with extensive CAA (Fig. 4).

FIGURE 3.

Double immunofluorescent labeling shows the endothelial localization of picalm. (A-F) Distribution of picalm in blood vessel walls closely follows that of the endothelial marker, factor VIII-related antigen/von Willebrand factor (FVIII) in sections of Alzheimer disease frontal cortex. (G-I) Human brain microvascular endothelial cells grown in culture express picalm and confirm the endothelial localization of this protein. Magnifications: ×350.

FIGURE 3.

Double immunofluorescent labeling shows the endothelial localization of picalm. (A-F) Distribution of picalm in blood vessel walls closely follows that of the endothelial marker, factor VIII-related antigen/von Willebrand factor (FVIII) in sections of Alzheimer disease frontal cortex. (G-I) Human brain microvascular endothelial cells grown in culture express picalm and confirm the endothelial localization of this protein. Magnifications: ×350.

FIGURE 4.

(A, B) Double immunofluorescent labeling for β-amyloid (Aβ) (A) and picalm (B) demonstrates good preservation of picalm even in vessels with extensive deposition of Aβ in cerebral amyloid angiopathy. (C) Merged image demonstrating different distribution of these 2 antigens. Magnifications: ×300.

FIGURE 4.

(A, B) Double immunofluorescent labeling for β-amyloid (Aβ) (A) and picalm (B) demonstrates good preservation of picalm even in vessels with extensive deposition of Aβ in cerebral amyloid angiopathy. (C) Merged image demonstrating different distribution of these 2 antigens. Magnifications: ×300.

Real-Time Polymerase Chain Reaction

Because picalm was found to be predominantly associated with endothelial cells, we quantified its gene expression in relation to 2 calibrator genes that encode for the endothelial cell markers FVIIIRA/VWF and CD31, and in relation to the housekeeping gene GAPDH. PICALM expression was significantly increased in the frontal cortex in AD in relation to all 3 calibrator genes (Fig. 5A), although the increase reached significance only in relation to GAPDH (Table 2; p = 0.047). There was no significant change in PICALM expression in AD compared with control temporal cortex or thalamus (Figs. 5B, C; Table 2).

FIGURE 5.

Levels of picalm messenger RNA in the frontal and temporal cortex and thalamus of Alzheimer disease (AD) and normal control brains was measured by real-time polymerase chain reaction. Fold change in PICALM expression in relation to GAPDH (•), FVIIIRA (▪;), and CD31 (♦) was calculated using the 2−ΔΔCt method. Expression is significantly increased in the AD frontal cortex in relation to GAPDH (p = 0.047) (A) but remains unchanged in the AD temporal cortex (B) and thalamus (C) versus normal controls. Graphs show geometric means and 95% confidence intervals.

FIGURE 5.

Levels of picalm messenger RNA in the frontal and temporal cortex and thalamus of Alzheimer disease (AD) and normal control brains was measured by real-time polymerase chain reaction. Fold change in PICALM expression in relation to GAPDH (•), FVIIIRA (▪;), and CD31 (♦) was calculated using the 2−ΔΔCt method. Expression is significantly increased in the AD frontal cortex in relation to GAPDH (p = 0.047) (A) but remains unchanged in the AD temporal cortex (B) and thalamus (C) versus normal controls. Graphs show geometric means and 95% confidence intervals.

TABLE 2.

Statistical Analysis of PICALM Expression in Alzheimer Disease and Control Groups In Relation to Calibrator Genes

Discussion

Picalm plays an important role in clathrin-mediated endocytosis, which is important in a number of processes (4, 5). Previous studies in cell lines and rat brain tissue reported its presence in neurons, astrocytes, and oligodendrocytes (4, 10, 11, 16). In addition, both overexpression and degradation of picalm disrupt the endocytic pathway (11, 13, 17). Our present studies show that picalm in human brain tissue is largely restricted to endothelial cells, with relatively little in neurons or glial cells. These ex vivo findings were supported by the demonstration of picalm in human cerebrovascular endothelial cells in vitro.

The transport of Aβ across the vessel wall and into the bloodstream is a major pathway of Aβ removal from the brain, impairment of which is thought to be important in the development of AD. The endothelial cell localization of picalm would be optimal for its participation in this process. In the small cohort we examined, PICALM expression was greater in the AD frontal cortex in comparison to all 3 genes evaluated, but the increase was significant only in relation to GAPDH (in relation to all cells) and not just endothelial cell markers. Because the analysis generated correlated rather than independent sets of data (as indicated by the similar alteration patterns in relation to the 3 calibrator genes), conventional correction for multiple testing (e.g. Bonferroni correction) would be excessively conservative, but the modest size of the increase should nonetheless be noted. Although our in vitro studies indicate that cerebrovascular endothelial cells are capable of producing picalm, it remains to be determined whether all of the picalm present in endothelial cells in the brain is synthesized within these cells.

The explanation for the elevated PICALM expression in the frontal cortex in AD is unclear. One possibility is that this is a response to elevated levels of Aβ. However, if that were the case, increased expression would also be expected in the temporal cortex. We speculate that other region-specific or biochemical changes related to the hyperphosphorylation of tau may modulate the influence of Aβ on PICALM expression. Alternatively, the timing and distribution of any change in PICALM expression may relate to the stage of spread of disease. PICALM overexpression can inhibit the formation of CCV (11); thus, too large an increase in expression in AD could be detrimental to the removal of Aβ by endocytosis. If the elevation in PICALM expression is a response to Aβ, a change in the ratio of picalm to Aβ beyond a certain limit could result in a negative feedback signal that reduces picalm levels.

PICALM variants are associated with late-onset AD risk (1, 2), but whether these variants influence the production of picalm protein and Aβ load has not yet been determined. Vessel wall uptake is not the only mechanism by which variations in PICALM might influence Aβ levels. Picalm may also affect the processing of APP. Amyloid precursor protein was found in CCV isolated from PC12 cells (6), and a reduction in APP internalization and in Aβ release was demonstrated in vitro in CHO cells (7) and in vivo in mouse brains after the disruption of clathrin-mediated endocytosis (8).

This is the first study to show the presence of picalm in endothelial cells of human brain tissue, and it suggests an increase in PICALM expression in AD. It remains unclear whether this increase is a reaction to the elevated level of Aβ or actually impairs Aβ uptake by endothelial cells and subsequent transport into the bloodstream. The elucidation of the role of picalm in AD may provide important insights into the pathogenesis of the disease.

References

1.
Harold
D
Abraham
R
Hollingworth
P
.
Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease
.
Nat Genet
 
2009
;
41
:
1088
93
2.
Lambert
J-C
Heath
S
Even
G
.
Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease
.
Nat Genet
 
2009
;
41
:
1094
99
3.
Dreyling
MH
Martinez-Climent
JA
Zheng
M
.
The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family
.
Proc Natl Acad Sci U S A
 
1996
;
93
:
4804
9
4.
Ford
MG
Pearse
BM
Higgins
MK
.
Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes
.
Science
 
2001
;
291
:
1051
55
5.
Marsh
M
McMahon
HT
.
The structural era of endocytosis
.
Science
 
1999
;
285
:
215
20
6.
Nordstedt
C
Caporaso
GL
Thyberg
J
.
Identification of the Alzheimer b/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells
.
J Biol Chem
 
1993
;
268
:
608
12
7.
Koo
EH
Squazzo
SL
.
Evidence that production and release of amyloid β-protein involves the endocytic pathway
.
J Biol Chem
 
1994
;
269
:
17386
89
8.
Cirrito
JR
Kang
J-E
Lee
J
.
Endocytosis is required for synaptic activity-dependent release of amyloid-β in vivo
.
Neuron
 
2008
;
58
:
42
51
9.
Kyriazis
GA
Wei
Z
Vandermey
M
.
Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: Implications for Alzheimer disease pathogenesis
.
J Biol Chem
 
2008
;
283
:
25492
502
10.
Yao
PJ
Zhang
P
Mattson
MP
.
Heterogeneity of endocytic proteins: Distribution of clathrin adaptor proteins in neurons and glia
.
Neuroscience
 
2003
;
121
:
25
37
11.
Tebar
F
Bohlander
SK
Sorkin
A
.
Clathrin assembly lymphoid myeloid leukemia (CALM) protein: Localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic
.
Mol Biol Cell
 
1999
;
10
:
2687
702
12.
Wu
F
Yao
PJ
.
Clathrin-mediated endocytosis and Alzheimer's disease: An update
.
Ageing Res Rev
 
2009
;
8
:
147
49
13.
Kim
JA
Kim
HL
.
Cleavage of purified neuronal clathrin assembly protein (CALM) by caspase 3 and calpain
.
Exp Mol Med
 
2001
;
33
:
245
50
14.
Chen
WJ
Goldstein
JL
Brown
MS
.
NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor
.
J Biol Chem
 
1990
;
265
:
3116
23
15.
Kirchhausen
T
Bonifacino
JS
Riezman
H
.
Linking cargo to vesicle formation: Receptor tail interactions with coat proteins
.
Curr Opin Cell Biol
 
1997
;
9
:
488
95
16.
Bushlin
I
Petralia
RS
Wu
F
.
Clathrin assembly protein AP180 and CALM differentially control axogenesis and dendrite outgrowth in embryonic hippocampal neurons
.
J Neurosci
 
2008
;
28
:
10257
71
17.
Rudinskiy
N
Grishchuk
Y
Vaslin
A
.
Calpain hydrolysis of α- and β2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration
.
J Biol Chem
 
2009
;
284
:
12447
58
18.
Heyman
A
Fillenbaum
GG
Mirra
SS
.
Consortium to Establish a Registry for Alzheimer's Disease (CERAD): Clinical, neuropsychological, and neuropathological components
.
Aging (Milano)
 
1990
;
2
:
415
24
19.
Consensus recommendations for the postmortem diagnosis of Alzheimer's disease
.
The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease
.
Neurobiol Aging
 
1997
;
18
:
S1
2
20.
Chalmers
K
Wilcock
GK
Love
S
.
APOE e4 influences the pathological phenotype of Alzheimer's disease by favouring cerebrovascular over parenchymal accumulation of Ab protein
.
Neuropathol Appl Neurobiol
 
2003
;
29
:
231
38
21.
Olichney
JM
Hansen
LA
Galasko
D
.
The apolipoprotein E epsilon 4 allele is associated with increased neuritic plaques and cerebral amyloid angiopathy in Alzheimer's disease and Lewy body variant
.
Neurology
 
1996
;
47
:
190
96
22.
Miners
JS
Baig
S
Tayler
H
.
Neprilysin and insulin-degrading enzyme levels are increased in Alzheimer disease in relation to disease severity
.
J Neuropathol Exp Neurol
 
2009
;
68
:
902
14
23.
Palmer
JC
Baig
S
Kehoe
PG
.
Endothelin-converting enzyme-2 is increased in Alzheimer's disease and up-regulated by Aβ
.
Am J Pathol
 
2009
;
175
:
262
70

Author notes

This study was supported by grants from the Dowager Eleanor Peel Trust and the Alzheimer's Society. This study was also supported by funding from the Medical Research Council for the Centre for Neuropsychiatric Genetic and Genomics (G0801418), by a MRC programme grant (G0300429), and by equipment grants from the Alzheimer's Research Trust and Bristol Research into Alzheimer's and Care of the Elderly. A Wellcome Trust Vacation Scholarship supported Sally Joseph's work on this project.