Medical

Communication

Biosci. Biotech. Res. Comm. 9(3): 366-370 (2016)

Deposition of monomeric C-reactive protein as

evidence of localised neurodegenerative diseases

Al Hsinawi M

and Al Baradie R*

King Khaled Majmaah Hospital, Al Majmaah, Kingdom of Saudi Arabia

Medical Laboratories Department, College of Applied Medical Sciences, Majmaah University, Al Majmaah,

Malek M Al-Hsinawi Director, King Khaled Majmaah Hospital Al Majmaah Kingdom of Saudi Arabia

Kingdom of Saudi Arabia

ABSTRACT

Monomeric-C-reactive protein (mCRP) is deposited in signi cant quantities within the brain parenchyma after stroke.

Since we have recently identi ed a possible role of this protein in supporting neurodegeneration and aberrant vascu-

lar development, we identi ed a small group of post-mortem brain samples from individuals who had AD and on his-

tological examination, evidence of tissue infarction/micro-infarction. Here we show that mCRP deposition is highest

in those regions affected by stroke or vascular disruption, and that within those same areas, there is more interaction

and co-localization between major classical proteins of neurodegeneration (-amyloid and tau. We hypothesise that

vascular disruption and concomitant release of mCRP within the brain tissue could exacerbate ongoing neurologi-

cal damage via stimulation of neuro-in ammation and from direct consequences of its action on both neuronal and

vascular health.

KEY WORDS: MONOMERIC C-REACTIVE PROTEIN; STROKE; ALZHEIMER’S DISEASE; NEURODEGENERATION; VASCULAR DEMENTIA

366

ARTICLE INFORMATION:

*Corresponding Author: raid5555@hotmail.com

Received 1

Sep, 2016

Accepted after revision 30

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

We have shown previously that monomeric C-reactive

protein (mCRP) was dramatically over-expressed in the

brain extracellular matrix (ECM) of patients following

acute ischaemic stroke (Slevin, 2010). 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 simi-

lar to that seen in a model of Alzheimer’s disease (AD)

(Tg x 3) ( Slevin, 2015).

mCRP also has been co-localised with CD105 in

microvessels suggesting angiogenesis. Phospho-arrays/

Western blotting identi ed signalling activation in both

Table 1: Patient information

Sample ID AGE SEX Braak-NFT CERAD Infarction

496T 84 F 2.3 - +

691F 83 M 3.4 4 +

697P 87 M 4.4 6 +

endothelial cells and neurons through p-IRS-1, p-Tau

and p-ERK1/2-which was blocked following pre-incu-

bation with mCRP-antibody suggesting that the anti-

body could have therapeutic potential. mCRP increased

vascular monolayer permeability and gap junctions,

increased NCAM expression and produced haemorrhagic

angiogenesis in mouse matrigel implants and clearly has

abnormal effects on the vascular system.

Previously, Strang et al (2012) have demonstrated

that A plaques generated in vitro were able to dissoci-

ate pCRP to mCRP, probably produced de-novo within

the brain ECM, whilst in vivo, more mCRP was identi-

 ed within frontal cortex regions in victims of AD than

normal control brains suggesting a possible pathological

or regulatory role in development of the disease. Cer-

ebrovascular disease, neurovascular dysfunction and

cerebral blood  ow abnormalities are now recognised

as critical in uences in the pathophysiological devel-

opment of AD, damaged, blocked or in patent vessels

having a severe effect on the function of local neuro-

vascular units (with approximately 80% of AD brains at

post-mortem shown to have signi cant vascular abnor-

malities), in addition to Vascular dementia, and lowers

the threshold for development particularly in younger

patients, (Toledo, 2013 and Montagne 2016).

Here, in the present study, we have investigated the

possible link between mCRP deposition and localization

within the brain and indicators of previous stroke or

vascular disruption and dementia.

MATERIAL AND METHODS

Samples of brain tissue were obtained from the Bristol

Brain Bank, where from a cohort of 10 individuals, 3

were identi ed as having concomitant histological evi-

dence of AD and stroke (assessed independently and also

by our clinical neurologist; Table 1), and a clinical reg-

istered history of AD. The AD cases all had a history of

progressive dementia and were selected on the basis of a

diagnosis according to CERAD of ‘de nite AD’ (Hyman

2012). AD neuropathological change was considered a

suf cient explanation for the dementia in all cases.

Paraf n processed sections from frontal parietal and

temporal regions were examined for expression of mCRP

and in addition co-staining (double immunohistochemi-

cal labelling with VIP-vector kits) for key marker pro-

teins of neurodegeneration (p-Tau and -amyloid).

Particular attention was given to analysis of staining

patterns associated with areas of tissue showing evi-

dence of in ammation, vascular damage and regions

showing morphological appearance of micro-infarct or

other previous stroke.

RESULTS AND DISCUSSION

The staining pattern revealed that mCRP was almost

absent from normal looking regions of brain tissue with-

out signs of neurodegeneration or previous tissue insult.

Occasional blood vessels, neurons and glia of brain tis-

sue showed positive mCRP staining. However, mCRP

expression increased in the areas demonstrating typical

AD pathology (i.e. amyloid deposits and neuro billary

tangles). In these later areas, mCRP was mainly localized

to blood vessels and neurons with a smaller expression

within glia. Since these areas were not associated with

previous infarction, the expression of mCRP is likely to

be de novo synthesis similar to that seen by Strang et al

[Strang 2012], however, within blood vessel walls, mCRP

could in ltrate from the circulation through damaged

intimal linings.

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 quanti cation of the extent of stain-

ing was not attempted. This is in line with our previ-

ously 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 [Slevin 2010].

B-amyloid staining and co-localization: Areas with

evidence of previous microinfarctions concomitantly

expressed more -amyloid. -amyloid deposition may

increase because of chronic in ammation after stroke

associated with chronic cerebrovascular dysfunction,

and this could be perpetuated at least in part by the

presence of excessive mCRP (Humpel 2011,Thiele, 2014

and McCaulley 2015).

Similarly, build-up of -amyloid is known to induce

neuro-in ammation directly, thus potentially perpetuat-

ing the neurodegenerative consequences (Hyman, 2012).

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS C-REACTIVE PROTEIN AS EVIDENCE OF LOCALISED NEURODEGENERATIVE DISEASES 367

Hsinawi and Baradie

Some of the amyloid plaques were close to adjacent

blood vessels where mCRP expression was abundant

(Figure 1B). Since mCRP is known to stimulate aber-

rant angiogenesis (Selvin, 2014), this expression could

have a potential negative in uence on existing or neo-

microvessel function and patency contributing to pro-

duction of a local hypo-perfusive neurodegenerative-

friendly environment.

The co-localization between mCRP and -amyloid in

blood vessels was also mainly seen within areas or adja-

cent to small infarcts (Figure 1B); however, it was also

observed sporadically in other areas of pure tau pathol-

ogy without clear infarction in the area.

-amyloid pathology within existing plaques was

mainly separate, with mCRP-positive ‘plaque’-like mate-

FIGURE 1. Single staining for mCRP-showing

increased expression within a micro-infarcted

area (496T; arrows; x 100)

FIGURE 2. Double labelled sections from previously infarcted regions of white (i) and grey

(ii) matter showing co-localization of -amyloid (red) with mCRP (blue-black) (691F; x

200; arrows).

FIGURE 3. Double staining for -amyloid and mCRP showing (i) -amyloid-positive (red) and mCRP-positive (blue-black)

separate plaques in none-infarcted regions (ii) a plaque containing both proteins within a previously infarcted area (arrows).

(697P; x 100 (inset x 200) (i) and x 200 (ii)).

rial generally being distinct from regular AD plaques, in

relatively normal looking tissue regions. However, adja-

cent to previously infarcted regions some of the plaques

(approximately 10% based on counting of 10 x  elds

of view at x 100 per section of 3 sections) contained a

combination of the two proteins (Figure 1C).

p-Tau staining and co-localization: The majority of

infarcted areas also exhibited other typical AD pathol-

368 C-REACTIVE PROTEIN AS EVIDENCE OF LOCALISED NEURODEGENERATIVE DISEASES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hsinawi and Baradie

ogy like Tau deposits. Most of the mCRP co-localized pri-

marily 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 num-

ber of neurons co-localising with the two proteins was

approximately (15% based on counting of 10 x  elds of

view at x 100 per section of 3 sections). Localization of

mCRP with phosphorylated tau in neurons could have

physiological signi cance.

Ourselves and others have demonstrated that mCRP

can phosphorylate tau (Ser 202,396) directly in vitro

(Selvin 2015; Guo 2015), possibly by a mechanism

involving GSK3. It is worthy of note that whilst co-

localising, the two proteins mCRP and p-Tau were pre-

sent generally at different positions within the neurons.

Toxic Tau  brils (neuro brillary tangles) were present

in some of the most degenerated regions and there was

evidence of co-localization of p-Tau with mCRP here

also suggesting as our previous work showed that mCRP

could contribute or perpetuate to their development

(arrows showing dark grey staining) potentially impli-

cating mCRP in its development (Figure E).

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 in uence of one process on

the other is subjective.

Given its strong aberrant biological properties associ-

ated with neurodegenerative signalling, vascular modu-

lation and angiogenesis, and its direct perpetuation of

in ammatory responses, a clear role for this protein in

promoting AD and VaD is proposed (Slevin, 2009). The

 ndings 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 pro-

cessing speed; and over 10% AD within 5 years (Desmond,

2002 and Mandzia, 2016), further research should study

potential mechanisms linking the two conditions with a

view to creating protective novel therapeutics.

CONCLUSION

mCRP is not found in normal-looking brain tissue of non

dementia patients, however it is produced and laid down

FIGURE 4. Double staining co-localization within micro-infarcted areas of p-tau (red) and mCRP (blue-black) (i) an iso-

lated double-labelled neuron (x 100; inset x 200) and (ii) neurodegenerative grey matter with separately stained neurons

for mCRP and p-Tau (arrows).

FIGURE 3. Double staining showing co-local-

ization of amyloid and mCRP (blue-black)-

NFTs-p-Tau (red) in an infarcted grey matter

brain region. (i) areas of co-localization are

shown by arrows (691T; x 200).

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS C-REACTIVE PROTEIN AS EVIDENCE OF LOCALISED NEURODEGENERATIVE DISEASES 369

370 C-REACTIVE PROTEIN AS EVIDENCE OF LOCALISED NEURODEGENERATIVE DISEASES BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hsinawi and Baradie

in large quantities within the brain following stroke, other

brain injury or conditions linked with neuro-in amma-

tion. We hypothesise that vascular disruption and con-

comitant release of mCRP within the brain tissue could

exacerbate ongoing neurological damage via stimulation

of neuro-in ammation and from direct consequences of

its action on both neuronal and vascular health.

DECLARATIONS-NONE

Abbreviations used: mCRP-Monomeric C-reactive

protein; VaD-Vascular dementia; AD-Alzheimer’s dis-

ease; ECM-Extra cellular matrix; CERAD-Consortium to

establish a registry for Alzheimer’s disease.

Ethics approval and consent to participate: Full ethi-

cal approval was obtained for the use of tissue sam-

ples as detailed by the South West Dementia Brain Bank

(SWDBB) and obtained from them.

Consent for publication: Has been obtained by all

authors

Availability of data and materials: N/A

Competing interests: The authors declare they have no

competing interests

Funding: This work was supported by the Research Cen-

tre of Healthcare Science at Manchester Metropolitan

University, and by Sheikh Abdullah bin Abdul Mohsen

Al-Tuwaijri project grants within, Majmaah University,

Saudi Arabia

ACKNOWLEDGEMENTS

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 assis-

tance in completing this piece of work.

REFERENCES

Desmond DW, Moroney JT,Sano M, Stern Y (2002), Incidence

of Dementia After Ischemic Stroke: Results of a Longitudinal

Study. Stroke. (33):2254-2262.

Hyman BT, Phelps CH, Beach TG, Bigo EH, Cairns NJ, Carrillo

MC, Dickson DW, Duyckaerts C, Frosch MP, Masliah E, Mirra

SS< Nelson PT, Scneider JA, Thal DR, Thies B, Trojanowski JQ,

Vinters HV, Montine TJ (2012) National Institute on Aging–

Alzheimer’s Association guidelines for the neuropathologic

assessment of Alzheimer’s disease. Alzheimer’s Dementia. 8(1):

1-13.

Guo H, Wang H, Wang C, Cheng Y, Zou Z, Li Y, Wu J, Xu J

(2015). C-Reactive Protein Induces Tau Hyperphosphorylation

via GSK3 Signaling Pathway in SH-SY5Y Cells. J Mol Neu-

rosci. 56(2):519-27.

Humpel C (2011) Chronic mild cerebrovascular dysfunction for

Alzheimer’s disease? Exp. Gerentol. 46(4): 225-232.

Mandzia JL, Smith EE, Horton M, Hanly P, Barber PA, God-

zwon C, Donaldson E, Asdaghi N, Patel S, Coutts SB (2016).

Imaging and Baseline Predictors of Cognitive Performance in

Minor Ischemic Stroke and Patients With Transient Ischemic

Attack at 90 Days. Stroke. 47(3):726-31.

McCaulley ME, Grush KA (2015) Alzheimer’s Disease: Explor-

ing the Role of In ammation and Implications for Treatment.

Int J Alzheimers Dis. Epub 2015 Nov 17.

Montagne A, Nation DA, Pa J, Sweeney MD, Toga AW, Zloko-

vic BV (2016) Brain imaging of neurovascular dysfunction in

Alzheimer’s disease. Acta Neuropathol. In press.

Slevin M, and Krupinski J (2009) A role for monomeric

C-reactive protein in regulation of angiogenesis, endothelial

cell in ammation and thrombus formation in cardiovascular

disease? Histol. Histopathol. (2009) (24)11: 1473-1478.

Slevin M, Matou-Nasri S, Turu M, Luque A, Rovira N, Badi-

mon L, Boluda S, Potempa L, Sanfeliu C, de Vera N, Krupinski

J (2010). Modi ed C-reactive protein is expressed by stroke

neovessels and is a potent activator of angiogenesis in vitro.

Brain Pathol. 20(1):151-65.

Slevin M, Matou S, Zeinolabediny Y Corpas R,Weston R,Liu

D,Boras E,Di Napoli M,Petcu E, Sarroca S Popa-Wagner A, Love,

S, Font MA, Potempa LA, Al-baradie R, Sanfeliu C, Revilla

S, Badimon L, and Krupinski J (2015). Monomeric C-reactive

protein-a key molecule driving development of Alzheimer’s

disease associated with brain ischaemia? Sci. Rep. 2015:

13281.

Strang F, Scheichl A, Chen YC, Wang X, Htun NM, Bassler

N, Eisenhardt SU, Habersberger J, Peter K (2012). Amyloid

plaques dissociate pentameric to monomeric C-reactive pro-

tein: a novel pathomechanism driving cortical in ammation in

Alzheimer’s disease? Brain Pathol. 22(3):337-46.

Thiele JR, Habersberger J, Braig D, Schmidt Y, Goerendt K,

Maurer V, Bannasch H, Scheichl A, Woollard KJ, von Dob-

schütz E, Kolodgie F, Virmani R, Stark GB, Peter K, Eisenhardt

SU (2014). Dissociation of pentameric to monomeric C-reactive

protein localizes and aggravates in ammation: In vivo proof

of a powerful proin ammatory mechanism and a new anti-

in ammatory strategy. Circulation. (1):35-50.

Toledo JB, Arnold SE, Raible K, Brettschneider J, Xie SX,

Grossman M, Kukull WA, Trojanowski JQ (2013) Contribution

of cerebrovascular disease in autopsy con rmed neurodegen-

erative disease cases in the National Alzheimer’s coordinating

centre. Brain. (136):2697-2706.