2017, Volume 33, Number 1, Page(s) 025-029
Expression of Monomeric C-Reactive Protein in Infarcted Brain Tissue from Patients with Alzheimer’s Disease
Mark SLEVIN1, Donghui LIU1, Glenn FERRIS1, Malik AL-HSINAWI2, Raid AL-BARADIE 2, Jerzy KRUPINSKI3
1Manchester Metropolitan University, School of Healthcare Science, Manchester, United Kingdom
2Department of Medical Laboratories, Majmaah University, Al Majma’ah, Saudi Arabia
3Department of Neurology, Hospital Universitari Mutua de Terrassa, Barcelona, Spain
Keywords: Monomeric C-reactive protein, Stroke, Alzheimer’s disease, Neurodegeneration, Vascular dementia
We have previously shown that monomeric-C-reactive protein is deposited in significant quantities within the brain parenchyma after
stroke. Since we have recently identified a possible role of this protein in supporting neurodegeneration and aberrant vascular development
we identified a small group of post-mortem brain samples from individuals who had Alzheimer’s disease and evidence of tissue infarction/
micro-infarction on histological examination.
Material and Method: We used immunohistochemistry staining to identify the monomeric-C-reactive protein expressed in the infarcted brain
Results: We showed that monomeric-C-reactive protein deposition was highest in those regions affected by stroke or vascular disruption, and
that within those same areas, there was more interaction and co-localization between major classical proteins of neurodegeneration (β-amyloid
Conclusion: We hypothesise that vascular disruption and concomitant release of monomeric-C-reactive protein within the brain tissue could
exacerbate ongoing neurological damage via stimulation of neuro-inflammation and from direct consequences of its action on both neuronal
and vascular cells.
We have previously shown that monomeric C-reactive
protein (mCRP) was dramatically over-expressed in the
brain extracellular matrix (ECM) of patients following
acute ischaemic stroke 1
. In addition, mCRP, unlike the
native pentameric molecule (pCRP), stimulated aberrant
angiogenesis in vitro, induced phosphorylation of tau by
neurons and tau-244-372 aggregation in vitro and following
injection into the hippocampus of mice, directly resulted
in cognitive and memory decline similar to that seen in a
model of Alzheimer’s disease (AD) (Tg x 3) 2
mCRP also co-localised with CD105 in microvessels
suggesting angiogenesis. Phospho-arrays/Western blotting
identified signalling activation in both endothelial cells and
neurons through p-IRS-1, p-Tau and p-ERK1/2-which was
blocked following pre-incubation with mCRP-antibody
suggesting that the antibody could have therapeutic potential.
mCRP increased vascular monolayer permeability and
gap junctions, increased NCA M expression and produced
haemorrhagic angiogenesis in mouse matrigel implants and
clearly has abnormal effects on the vascular system.
Previously, Strang et al. 3 demonstrated that Aβ plaques
generated in vitro were able to dissociate pCRP to mCRP
probably produced de-novo within the brain ECM, whilst
in vivo, more mCRP was identified within frontal cortex
regions in victims of AD than normal control brains
suggesting a possible pathological or regulatory role in
development of the disease.
Cerebrovascular disease, neurovascular dysfunction and
cerebral blood flow abnormalities are now recognised as
critical influences in the pathophysiological development of
AD, damaged, blocked or in patent vessels having a severe
effect on the function of local neurovascular units (with
approximately 80% of AD brains at post-mortem shown to
have significant vascular abnormalities).
Damage to the deep penetrating vessels within the brain
such as following lacunar stroke, ischaemic stroke or
head trauma, may lower the threshold for development
of vascular and other (mixed) dementia, particularly in
younger patients 4, 5.
Here, we investigated the possible link between mCRP
deposition and localization within the brain and indicators
of previous stroke or vascular disruption and dementia.
Samples of brain tissue were obtained from the Bristol
Brain Bank, where from a cohort of 10 individuals
3 were identified as having concomitant histological
evidence of AD and stroke (assessed independently and
also by our clinical neurologist; Table I
), and a clinical
registered history of AD. The AD cases all had a history of
progressive dementia and were selected on the basis of a
diagnosis according to Consortium to establish a registry
for Alzheimer’s disease (CERAD ) of ‘definite AD’ 6
AD neuropathological change was considered a sufficient
explanation for the dementia in all cases.
Full ethical approval was obtained for the use of tissue
samples as detailed by the South West Dementia Brain
Bank (SWDBB) and obtained from them.
Mouse monoclonal antibody to human mCRP sub-unit
(M8C10) was obtained from Dr L.A, and was stated as
previously 2. Mouse monoclonal antibodies to p-Tau
and β-amyloid were purchased from Abcam (UK).
Standard Immunohistochemistry staining was carried
out according to the protocols and fully characterized as
described previously 1. Paraffin processed sections from
frontal, parietal and temporal regions were examined for
expression of mCRP (mouse monoclonal; 1:10 dilution;
blue-grey colour). These were our own characterized
antibodies, and originally a gift from Dr Larry Potempa
Roosevelt University, USA 1. In addition co-staining
(double immunohistochemical labelling for key marker
proteins of neurodegeneration (rabbit polyclonal p-Tau
and β-amyloid; used at 1:500 dilution; brown) with VIP
VECTA STAIN -ELITE-vector HRP kits (from Vector labs.
com) was carried out. Particular attention was given to
analysis of staining patterns associated with areas of tissue
showing evidence of inflammation, vascular damage and
regions showing morphological appearance of microinfarct
or other previous stroke.
Staining patterns and intensity were observed by an
independent neuropathologist in a blinded fashion. Total
staining was not quantified but overall expression in
different regions and by specific recognisable cell types
over the whole region were characterised using our double
size sections. For plaque numbers and numbers of neurons
positively stained, counting of 10 × fields of view at × 100
per section of three sections was employed. A distinction
between strong (intense) and weak staining was also made.
| Immunohistochemical Findings
The staining pattern revealed that mCRP was almost absent
from normal looking regions of brain tissue without signs
of neurodegeneration or previous tissue insult. Occasional
blood vessels, neurons and glia of brain tissue showed
positive mCRP staining. However, mCRP expression
increased in the areas demonstrating typical AD pathology
(i.e. amyloid deposits and neurofibrillary tangles). In these
later areas, mCRP was mainly localized to blood vessels and
neurons with a smaller expression within glia.
mCRP expression was most abundantly present in areas
of microinfarction (and adjacent regions) in both grey
and white matter, whilst in relatively normal looking
areas, expression was weak or none-existent (Figure 1A).
mCRP expression was not seen in normal looking regions
and hence quantification of the extent of staining was not
Β-amyloid staining and co-localization: Areas with
evidence of previous microinfarctions concomitantly
expressed more β-amyloid. Some of the amyloid plaques
were close to adjacent blood vessels where mCRP expression
was abundant (Figure 1B). The co-localization between
mCRP and β-amyloid in blood vessels was also mainly
seen within areas or adjacent to small infarcts (Figure 1B);
however, it was also observed, sporadically in other areas
of pure tau pathology without clear infarction in the area.
β-amyloid pathology within existing plaques was mainly
separate, with mCRP-positive ‘plaque’-like material
generally being distinct from regular AD plaques, in
relatively normal looking tissue regions. However, adjacent
to previously infarcted regions some of the plaques
(approximately 10% based on counting of 10 × fields of
view at × 100 per section of three sections) contained a
combination of the two proteins (Figure 1C).
Click Here to Zoom
|Figure 1: A) Single staining for monomeric C-reactive protein (mCRP)-showing increased expression within a micro-infarcted area (brown, arrows) (mCRP; x100), B) Double labelled sections from previously infarcted regions of white (i) and grey (ii) matter showing co-localization of β-amyloid (blue-grey) with mCRP (brown) (IHC; x200), C) Double staining for β-amyloid and mCRP showing (i) β-amyloid-positive (brown) and mCRP-positive (blue-grey) separate plaques in none-infarcted regions (ii) a plaque containing both proteins within a previously infarcted area (arrows) (IHC; x100) (inset x 200) (i) and x 200 (ii). D) Double staining co-localization within micro-infarcted areas of p-tau (brown) and mCRP (blue-grey) (i) an isolated double-labelled neuron (IHC; x100; inset x 200) and (ii) neurodegenerative grey matter with separately stained neurons for mCRP (blue-grey) and p-Tau (arrows). E) Double staining showing co-localization of amyloid and mCRP (blue-grey)-NFTs-p-Tau (brown) in an infarcted grey matter brain region. (i) areas of colocalization are shown by arrows (IHC; x200).
p-Tau Staining and Co-Localization: The majority of
infarcted areas also exhibited other typical AD pathology
like tau deposits. Most of the mCRP co-localized primarily
with tau within neurons in peri-nuclear regions (Figure 1D), although, again, the majority of neurons were either
p-Tau or mCRP positive however the number of neurons
co-localising with the two proteins was approximately (15%
based on counting of 10 × fields of view at × 100 per section
of three sections). Co-localisation of the two proteins
mCRP and p-Tau was present but generally at different
positions within the neurons. (arrows showing dark grey
staining) (Figure 1E).
Toxic Tau fibrils (neurofibrillary tangles) were present
in some of the most degenerated regions and there was
evidence of co-localization of p-tau. Limitations of this
‘case study’ analysis are clearly from the small numbers
of sections and patients reviewed. In addition, the relative
timing of events in these patients e.g. infarction versus AD
symptoms is not known and so the relative influence of one
process on the other is subjective.
Significant expression of mCRP was found adjacent to
infarcted areas. This is in line with our previously published
observation and hypothesis of a direct link between mCRP
deposition and vascular damage in the brain linking it to
subsequently and chronically hypo-perfused tissue regions
. Since mCRP was also found in smaller quantities
in areas that were not directly associated with previous
infarction, the expression of mCRP is likely to be due to
de novo synthesis similar to that seen by Strang et al 3
however, within blood vessel walls, mCRP could infiltrate
directly from the circulation through damaged intimal
linings in patients with vascular disease.
β-amyloid deposition may increase in damaged areas
because of chronic inflammation after stroke associated
with chronic cerebrovascular dysfunction, and this could
be perpetuated at least in part by the presence of excessive
mCRP 7-9. Similarly, build-up of β-amyloid is known
to induce neuro-inflammation directly, thus potentially
perpetuating the neurodegenerative consequences 6.
Since mCRP is known to stimulate aberrant angiogenesis
2, this expression could have a potential negative influence
on existing or neo-microvessel function and patency
contributing to production of a local hypo-perfused
Localization of mCRP with phosphorylated tau in neurons
could have physiological significance. Others and ourselves
have demonstrated that mCRP can phosphorylate tau (Ser
202, 396) directly in vitro 2, 10, possibly by a mechanism
involving GSK3β. It is worthy of note that whilst colocalising
the two proteins mCRP and p-Tau were present
generally at different positions within the neurons.
However, this still suggests that mCRP could contribute to
abnormal activation of these neurons after stroke 2.
In summary, mCRP was not found in normal-looking
brain tissue of none dementia patients, however it is
produced and laid down in large quantities within the
brain following stroke, other brain injury or conditions
linked with neuro-inflammation. Given its strong aberrant
biological properties associated with neurodegenerative
signalling, vascular modulation and angiogenesis, and its
direct perpetuation of inflammatory responses, a clear role
for this protein in promoting AD and VaD is proposed 11.
The findings shown here strengthen this hypothesis further
providing case studies of AD patients where vascular
insults probably linked to a local hypoxic environment
appear to be highlighted by a strong deposition of mCRP
and concomitant disproportionate evidence of localised
neurodegenerative disease. Since the majority of patients
who suffer serious stroke go on to develop cognitive decline
lower executive function, and psychomotor processing
speed; and over 10% AD within 5 years 12, 13, further
research should study potential mechanisms linking the
two conditions with a view to creating protective novel
CONFLICT of INTEREST
The authors declare no conflict of interest.
SOURCE of FUNDING
This work was supported by the Research Centre of
Healthcare Science at Manchester Metropolitan University
and by Sheikh Abdullah bin Abdul Mohsen Al-Tuwaijri
project grants within, Majmaah University, Saudi Arabia.
We would like to thank the South West Dementia Brain
Bank (SWDBB) for providing brain tissue for this study.
The SWDBB is part of the Brains for Dementia Research
programme, jointly funded by Alzheimer’s Research UK
and Alzheimer’s Society and is supported by BRACE (Bristol
Research into Alzheimer’s and Care of the Elderly) and the
Medical Research Council. This research was supported
by Sheikh Abdullah bin Abdul Mohsen Al-Tuwaijri
project grants within, Majmaah University, Saudi Arabia.
The authors would like to express their gratitude towards
Sheikh Abdullah Abdul Mohsen Al-Tuwaijri, Rector Dr.
Khalid Saad Al Muqrin for providing the necessary support
and assistance in completing this piece of work.
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