WO2018042182A1

WO2018042182A1 - Compositions and uses thereof

Info

Publication number: WO2018042182A1
Application number: PCT/GB2017/052541
Authority: WO
Inventors: Mark Slevin; Johannes Boltze; Lawrence A. Potempa
Original assignee: Manchester Metropolitan University
Priority date: 2016-09-02
Filing date: 2017-09-01
Publication date: 2018-03-08
Also published as: GB201614897D0

Abstract

The present invention relates to compositions for use in the treatment, prevention or management of neurological degeneration or dementia, comprising inhibitors or antagonists of monomeric C-reactive protein (mCRP), and optionally, the inhibitor or antagonist modulates the activity ofthe cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP.

Description

COMPOSITIONS AND USES THEREOF

Technical Field of the Invention

The present invention relates to compositions, such as antibodies, which are capable of binding to monomeric C-reactive protein (mCRP), in particular to the cholesterol binding region (CBR) and/or optionally to the C-terminal octapeptide region of mCRP, so as to modulate its activity. Such compositions are particularly suited for use in the prevention, management and/or treatment of diseases associated with neurological degeneration.

Background to the Invention

Certain neurodegenerative diseases are related to or involve inflammation. The inflammation may be as a result of neurovascular or traumatic events or due to acute or chronic hypoperfusion.

Thrombotic or embolic vessel occlusion or haemorrhage can result in local inflammation. Strokes are typically caused by an ischaemic event in the brain, and have been shown to increase the risk of neurodegenerative conditions such as dementia or Alzheimer's disease by four to twelve (4 to 12) fold. The present inventors have unexpectedly found that the monomeric C-reactive protein (mCRP) isoform is deposited in significant amounts in the brain following an ischaemic event.

mCRP is a monomer of native pentameric CRP. Pentameric CRP is an acute phase pentraxin produced mainly in the liver in response to infection. Its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells, stimulating the activation of the complement system by the C1Q complex. In contact with cells and/or tissue it converts to the monomeric form, i.e. mCRP. As such, mCRP is typically found in regions of tissue injury where it can remain chronically with the extracellular matrix and cells.

It is an object of the present invention to provide a composition which can be used to reduce neurological degeneration or dementia brought on by cerebrovascular impairment, for example by an ischaemic or traumatic event or hypoperfusion. In particular, it is an object of the present invention to provide a therapy which targets mCRP for the prevention, management, mitigation or treatment of neurodegenerative conditions. Summarv of Invention

The invention is defined in the appended claims and also includes the combination of the aspects and preferred features hereinafter described except where such a combination is clearly impermissible or expressly avoided.

In accordance with an aspect of the present invention, there is provided a composition for use in the treatment, prevention or management of neurological degeneration or dementia, comprising an inhibitor or antagonist of monomeric C-reactive protein (mCRP).

In a related aspect, there is provided a composition for use in a method of treatment, prevention or management of neurological degeneration or dementia, comprising administering an inhibitor or antagonist of mCRP to an individual.

In an additional related aspect, there is provided a composition for use in a method of preventing neurological degeneration or dementia, comprising administering an inhibitor or antagonist of mCRP to an individual who: (i) has a history of ischaemic events; (ii) is experiencing an ischaemic event; (iii) has experienced an ischaemic event within the last 72 hours; or (iv) has elevated levels of mCRP.

In another related aspect, there is provided a composition comprising an inhibitor or antagonist of monomeric C-reactive protein mCRP for use as a medicament.

In yet another related aspect, there is provided the use of a composition comprising an inhibitor or antagonist of monomeric C-reactive protein mCRP for the manufacture of a medicament for the treatment, prevention or management of neurological degeneration or dementia.

The terms "monomeric C-reactive protein" and "mCRP" are intended to encompass related forms of mCRP such as the disulphide-bond reduced form of mCRP (r_mCRP) or the mutated protein, and in particular, a protein where the cysteine residues in mCRP have been substituted with another amino acid (such as alanine residues) (e.g. C36A; C97A).

The term "neurological degeneration or dementia" is intended to cover a range of degenerative neurological conditions including, but not limited to, neuronal degeneration, vascular dementia, cerebral amyloid angiopathy, oxidative inflammatory damage to cerebral microvessels, and Alzheimer's disease.

The term "management" is intended to cover maintaining the current state of disease, and/or or reducing the severity, seriousness, or symptoms of the disease or related complications thereof.

The inhibitor or antagonist of the above aspects may modulate the activity of the cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP. The inhibitor or antagonist may bind to epitopes expressed on mCRP and in particular bind to amino acid residues of mCRP at amino acid residues of about 35 to about 47 and/or epitopes expressed on mCRP and in particular bind to amino acid residues of mCRP at amino acid residues of about 199 to about 206.

The inhibitor or antagonist or the composition may bind to at least part of or a fragment of the amino acid sequence VCLH FYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% or 85% homology thereof. More preferably, the inhibitor or antagonist or the composition may bind to at least part of or a fragment of the amino acid sequence VCLH FYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 90% homology thereof. Even more preferably, the inhibitor or antagonist or the composition may bind to at least part of or a fragment of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP of mCRP (SEQ ID No. 3) or derivative sequences having at least 95% homology thereof. Most preferably, the inhibitor or antagonist or the composition binds to at least part or a fragment of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP.

The inhibitor or antagonist or the composition may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% or 85% homology thereof. More preferably, the inhibitor or antagonist or the composition may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 90% homology thereof. Even more preferably, the inhibitor or antagonist or the composition may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP of mCRP (SEQ ID No. 3) or derivative sequences having at least 95% homology thereof. Most preferably, the inhibitor or antagonist or the composition binds to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP.

The inhibitor or antagonist may comprise a peptidic moiety according to a Formula 1 or salt, derivative, prodrug or mimetic thereof:

Formula V. [X1-X2-X3-X4-X5-X6] wherein X1 may be present or absent, when X1 is present, X1 comprises V; wherein X2 may be present or absent, when X2 is present, X2 comprises C; wherein X3 comprises L; wherein X4 comprises H, R, G, W or Y; wherein X5 comprises up to 170 amino acids; and wherein X6 may be present or absent, when X6 is present, X6 comprises

FTKPQLWP (SEQ ID No. 3).

X5 may comprise C at position 59 if X2 is present.

X1 to X5 may comprise VCLHFYTELSSTR (SEQ ID No. 2).

The table below shows the single letter amino acid and three letter amino acid codes:

The inhibitor or antagonist may comprise a peptidic moiety according to a Formula 2 or salt, derivative, prodrug or mimetic thereof:

Formula 2: [l_/V-X(1-5)-Y-X(1-5)-R/K] The composition may comprise amino acid residues or amino acid analogues.

The composition may comprise a peptide or peptide mimetic molecule. A peptide memetic may be a small molecule showing similar binding characteristics to the peptide it is mimicking.

The inhibitor or antagonist may comprise an antibody or antibody mixture.

The inhibitor or antagonist may be artificially generated. That is to say that it is not naturally occurring. The inhibitor or antagonist may however be a naturally occurring molecule whose concentration and formulation in a medicament enables it to be used for the treatment, prevention or management of neurological degeneration or dementia, whereas otherwise it would have no or limited efficacy.

The targeted mCRP may be from any suitable source. In certain embodiments, the targeted mCRP may be derived from a human or other mammalian source. Preferably, the mCRP may be from a human source. More preferably, the mCRP may be human mCRP represented by SEQ ID No: 1. For the avoidance of doubt, human mCRP has 206 amino acids.

The neurological degeneration or dementia may be related to or due to inflammation. The inflammation may be the result of neurovascular or traumatic event or to acute or chronic hypoperfusion.

The neurological degeneration or dementia may be related to or due to thrombotic or embolic vessel occlusion.

The neurological degeneration or dementia may be related to or due to an ischaemic event.

In another related aspect of the invention, there is provided a method of treating, preventing or managing a neurological degeneration or dementia in an individual, comprising the steps: (i) removing a portion of circulating blood from an individual who is affected by or at risk of neurological degeneration or dementia; (ii) substantially removing monomeric C- reactive protein (mCRP) from the blood portion or rendering monomeric C-reactive protein (mCRP) inactive in the portion of blood; and (iii) returning the portion of blood to the individual.

It will of course be apparent that the method of treatment could employ a dialysis machine or similar apparatus for removing and treating the blood before returning it to an individual.

In a further related aspect, there is provided an antibody or antibody mixture, for use in the treatment, prevention or management of neurological degeneration or dementia, wherein the antibody or antibody mixture is capable of binding to the cholesterol binding region (CBR) and optionally to the C-terminal octapeptide region of mCRP.

In another related aspect, there is provided an antibody or antibody mixture, for use in a method of treatment, prevention or management of neurological degeneration or dementia, wherein the antibody or antibody mixture is capable of binding to the cholesterol binding region (CBR) and optionally to the C-terminal octapeptide region of mCRP. ln certain preferred embodiments, the antibody binds to the cholesterol binding region of human mCRP. The cholesterol bonding region of human mCRP is from amino acids 35 to 47. The cholesterol binding region of human mCRP is represented by SEQ ID NO: 2. For the avoidance of doubt, the cholesterol binding region is located in the cholesterol binding domain of mCRP which includes amino acid residues that may form an inter-chain disulphide bond. In human mCRP, amino acids 36 and 97 may form an inter-chain disulphide bond. The antibody of the present invention may bind to the cholesterol binding region of mCRP when the cholesterol binding domain of said mCRP has a disulphide bond, such as between amino acids 36 and 97, or when the cholesterol binding domain of said mCRP is in the reduced form. Preferably, antibody of the present invention may bind to the cholesterol binding region of mCRP when the cholesterol binding domain of said mCRP is in the reduced form. Advantageously, it has surprisingly been found by the present inventors that the antibody of the present invention is able to bind more strongly to the cholesterol binding region of mCRP when the cholesterol binding domain of said mCRP is in the reduced form.

The antibody or antibody mixture of the present invention may optionally bind to the C- terminal octapeptide region of mCRP. In certain preferred embodiments, the antibody may optionally bind to the C-terminal octapeptide region of human mCRP. The C-terminal octapeptide region of human mCRP is from amino acids 199 to 206. The C-terminal octapeptide region of human mCRP is represented by SEQ ID NO: 3.

In certain embodiments, the antibody binds to the cholesterol binding region of human mCRP which is located at amino acids 35 to 47 and is represented by SEQ ID NO: 2.

In certain embodiments, the antibody binds to the cholesterol binding region of human mCRP which is located at amino acids 35 to 47 and is represented by SEQ ID NO: 2 and to the C-terminal octapeptide region of human mCRP which is located at amino acids 199 to 206 and is represented by SEQ ID NO: 3.

The antibody or antibody mixture may bind to at least part of or a fragment of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% or 85% homology thereof. More preferably, the antibody or antibody mixture may bind to at least part of or a fragment of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 90% homology thereof. Even more preferably, the antibody or antibody mixture may bind to or a fragment of at least part of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP of mCRP (SEQ ID No. 3) or derivative sequences having at least 95% homology thereof. Most preferably, the antibody or antibody mixture binds to or a fragment of at least part of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP.

The antibody or antibody mixture may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% or 85% homology thereof. More preferably, the antibody or antibody mixture may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 90% homology thereof. Even more preferably, the antibody or antibody mixture may bind to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP of mCRP (SEQ ID No. 3) or derivative sequences having at least 95% homology thereof. Most preferably, the antibody or antibody mixture binds to the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP.

An antibody may be obtained by any suitable method. Suitable methods will be well known to a person skilled in the art. For example, the antibody may be obtained by using natural mCRP as an immunising antigen, may be obtained using recombinant mCRP as an immunising antigen or may be obtained using a peptide comprising the amino acid sequence of the binding region, such as, for example, amino acids 35 to 47 of human mCRP and optionally amino acids 199 to 206 of human mCRP, as an immunising antigen. Preferably, the antibody may be obtained using recombinant mCRP as an immunising antigen, more preferably using recombinant human mCRP as an immunising antigen. In certain embodiments, the antibody may be obtained using recombinant mCRP, such as human mCRP, having a wild type sequence or having a sequence which comprises one or more mutations as an immunising antigen. Preferably, the antibody may be obtained using recombinant mCRP, such as human mCRP, having a sequence which comprises one or more mutations as an immunising antigen. More preferably, the antibody may be obtained using recombinant human mCRP having a sequence which comprises mutations at amino acid residues 36 and/or 97, most preferably at amino acid residues 36 and 97 as an immunising antigen. For example, amino acid residues 36 and/or 97 may be mutated from cysteine to serine.

Advantageously, the use of recombinant human mCRP having a sequence which comprises mutations from, for example, cysteine to serine at amino acid residues 37 and/or 97 exposes the cholesterol binding site because there is no disulphide bridge in the cholesterol binding domain.

For the avoidance of doubt, by 'natural mCRP' is meant that the mCRP is obtained from a natural source, i.e. is expressed endogenously. For the avoidance of doubt, by 'recombinant mCRP' is meant that the mCRP is expressed heterologously in, for example, in a genetically modified or engineered organism comprising genetic material which has been artificially constructed an inserted into the organism. The genetic material may comprise endogenous or heterologous nucleic acids which may or may not have been further genetically modified.

In certain embodiments, when a peptide comprising the amino acid sequence of the binding region, such as, for example, amino acids 35 to 47 of human mCRP and optionally amino acids 199 to 206 of human mCRP, is used as an immunising antigen, the peptide may be bonded to a carrier. For example, the carrier may be a mammal-derived protein such as albumin or globulin, a protein such as keyhole limpet hemocyanin, a microorganism such as inactivated tubercle bacillus or a polyamino acid such as polylysine or polyasparagine.

Recombinant mCRP may be produced by any suitable method. For example, recombinant mCRP may be produced using a genetic engineering method or a peptide synthesis method. Preferably, recombinant mCRP may be produced using a genetic engineering method. More preferably, recombinant mCRP may be produced using a genetically modified or engineered organism, such as, for example, Escherichia coli (E.coli). Methods of genetically engineering or modifying organisms such as E.coli will be known to a person skilled in the art, for example, by cloning into expression vectors a polynucleotide encoding the mCRP peptide amino acid sequence to obtain a recombinant plasmid, transforming a host organism, such as E.coli, with the obtained recombinant plasmids, culturing the transformants and causing expression of the polynucleotide encoding mCRP and extracting and purifying the expressed recombinant mCRP from the culture. The host organism, such as E.coli, may be genetically modified or engineered to comprise endogenous or heterologous nucleic acids for the expression of mCPR, such as human mCRP.

The antibody of the present invention may be polyclonal or may be monoclonal.

In certain embodiments, the antibody of the present invention may be monoclonal. The monoclonal antibodies may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies may be obtained using hybridoma technology, such as fusing antibody producing cells of antigen- immunised mammals with mammalian myeloma cells to produce a hybridoma cell line. Preferably, the hybridoma cell line is produced by first immunising a mammal with an immunising antigen to produce an immunised mammal. Suitable immunising antigens are as defined above. The mammal may be immunised by any suitable method such as, for example, by intraperitoneal, subcutaneous, intravascular, intramuscular or intrasplenic injection or by oral administration. Preferably, the immunising antigen may be administered as a suspension or solution in a buffer, such as phosphate buffered saline (PBS), optionally with an adjuvant, such as Freund's complete adjuvant. Any suitable mammal may be used such as, for example, mice, rats, rabbits, sheep or goats. It will be appreciated by a person skilled in the art that the mammal should typically be chosen so as to be compatible with the myeloma cells used in the subsequent cell fusion step. Preferably, the immunising antigen is administered to the mammal several times, such as 2, 3, 4 or more times, at 4 to 21 day intervals. Typically, the antibody producing cells of the immunised mammal may be splenic cells. Preferably, the splenic cells of the immunised mammal may be collected and fused with mammalian myeloma cells. The mammalian myeloma cells may be from any suitable source such as, for example, mice, rats or rabbits. Preferably, the mammalian myeloma cells may be from the same mammalian source as the mammal immunised with the immunising antigen. Preferably, the myeloma cells may be from mice. Preferably, the mammalian myeloma cells are selected so as to have a hypoxyanthine-guanine- phosphoribosyltransferase deficiency (HGPRT) and/or a thymidine kinase deficiency (TK^"). For example, the mammalian myeloma cells may be mouse P3/NS1/1-Aq4-1 cells. The splenic cells may be fused to the mammalian myeloma cells by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the splenic cells may be fused to the mammalian myeloma cells using electrofusion, optionally in the presence of a fusion promoter such as, for example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ). Preferably, the splenic cells and the mammalian myeloma cells may be mixed at a ratio of 1 : 1 to 10: 1.

Typically, the fused cells may be cultured and screened to selectively obtain hybridomas. For the avoidance of doubt, a 'hybridoma' is a hybrid cell which is able to produce the antibodies of the invention. The fused cells may be cultured in any suitable medium. The fused cells may be screened for hybridomas by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the fused cells may be screened for hybridomas using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR). Preferably, the fused cells may be screened for hybridomas using enzyme-linked immunosorbent assay (ELISA). It will be appreciated by a person skilled in the art that it is the supernatant of the culture of the fused cells that is typically screened. Preferably, the fused cells may be screened for hybridomas by screening for binding to the cholesterol binding region and optionally the C-terminal octapeptide region of mCRP, such as human mCRP. More preferably, the fused cells may be screened for hybridomas by screening for binding to the cholesterol binding region and optionally the C-terminal octapeptide region of mCRP, such as human mCRP, using enzyme-linked immunosorbent assay (ELISA).

The monoclonal antibodies may be produced using the obtained hybridomas using any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies may be produced by culturing the obtained hybridomas in a suitable medium or in the abdominal cavities of a suitable mammal such as, for example, a mouse. Preferably, the monoclonal antibodies may be produced by culturing the obtained hybridomas in a suitable medium. Suitable media will be known to a person skilled in the art. It will be appreciated by a person skilled in the art that the medium should be chosen so as to be compatible with hybridoma culture. For example, the monoclonal antibodies may be produced by culturing the obtained hybridomas in RPMI 1640 medium containing foetal bovine serum, L-glutamine, L-pyruvic acid and/or antibiotics such as penicillin or streptomycin. Any suitable amount of the obtained hybridomas may be added to the medium. For example, about 10¹ to 10¹⁰ individual hybridomas, preferably about 10² to 10⁶ individual hybridomas, more preferably about 10⁴ to 10⁵ individual hybridomas, may be added per milli litre (ml) of medium. The culture may be performed under any suitable conditions. For example, the culture may be performed at a carbon dioxide (C0₂) concentration of about 1 to 10%, preferably about 5%, a temperature of about 30 to 40°C, preferably 37°C, for a time of about 0.5 to 7 days, preferably 1 to 4 days.

The monoclonal antibodies produced by the cultured hybridomas may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies produced by the cultured hybridomas may be obtained by centrifugation of the supernatant of the culture.

In certain alternative embodiments, when the monoclonal antibodies are produced by culturing the obtained hybridomas in the abdominal cavities of a suitable mammal such as, for example, a mouse, the obtained hybridomas may be intraperitoneally administered to the mammal such as, for example, mouse. The monoclonal antibodies produced by the culture in the abdominal cavities of a suitable mammal such as, for example, a mouse may then be obtained by collecting the fluid in the peritoneal cavity.

The monoclonal antibodies produced by culturing the obtained hybridomas in a suitable medium or in the abdominal cavities of a suitable mammal such as, for example, a mouse may be used directly or may be purified. Preferably, the monoclonal antibodies may be purified. The monoclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the monoclonal antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.

In certain embodiments, the antibody of the present invention may be polyclonal. The polyclonal antibodies may be produced by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be produced by immunising a mammal with an immunising antigen to induce the production of antibodies specific for the said immunising antigen. Suitable immunising agents are as defined above. Any suitable mammal may be used such as, for example, mice, rats or rabbits. The polyclonal antibodies produced by the immunised mammal may be obtained by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be obtained by collecting the serum of the immunised mammal.

The polyclonal antibodies produced by immunising a mammal with an immunising antigen and collecting the serum of the immunised mammal may be used directly or may be purified. Preferably, the monoclonal antibodies may be purified. The polyclonal antibodies may be purified by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may be purified by ammonium sulphate precipitation, ion exchange chromatography or an anti-lgG antibody column.

The polyclonal antibodies may be screened for binding to the cholesterol binding region and optionally the C-terminal octapeptide region of mCRP using any suitable method. Suitable methods will be known to a person skilled in the art. For example, the polyclonal antibodies may screened for binding to the cholesterol binding region and optionally the C- terminal octapeptide region of mCRP using enzyme immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or surface plasmon resonance (SPR). Preferably, the polyclonal antibodies may screened for binding to the cholesterol binding region and optionally the C-terminal octapeptide region of mCRP using enzyme- linked immunosorbent assay (ELISA).

Preferably, the antibody of the present invention is monoclonal.

The antibody of the present invention may be any suitable isotype. Preferably, the antibody is of isotype IgG. For the avoidance of doubt, antibodies of the isotype IgG typically comprise four peptide chains, of which two are heavy chains and two are light chains, and have two fragment antigen-binding (Fab) regions. The Fab regions comprise complementary determining regions (CDRs) which are the part of the antibody which bind to the antigen.

The antibody of the present invention may be a whole antibody, an antibody fragment or a modified form thereof. Suitable examples of whole antibodies include, but are not limited to, monovalent or divalent antibodies. Suitable examples of antibody fragments include, but are not limited to, Fab, F(ab')₂, Fv, Fab/c having one Fab and complete Fc, and single chain Fv (scFv) having heavy (H) or light (L) chain Fvs connected by a suitable linker.

In certain embodiments, the antibody of the present invention may be optimised or may be humanised. 'Optimised', and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, so as to be suitable for use in the patient to which it is to be administered. 'Humanised', and like terms as used herein, means that the amino acid sequence of the antibody is adapted, such as by mutation or modification including, for example, glycosylation, to reduce the composition of non-human amino acid sequences in the antibody.

The antibody of the present invention, when humanised, may be partially humanised or may be substantially fully humanised. By 'partially humanised' is mean that part of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody. The antibody, when partially humanised, may be partially humanised in any region of the antibody. Preferably, the antibody, when partially humanised, may be partially humanised in one or more of the variable fragment antigen-binding (Fab) regions of the antibody. By 'substantially fully humanised' is meant that substantially all of the amino acid sequence of the antibody has been adapted, such as by mutation or modification including, for example, glycosylation, to be the same as the amino acid sequence of the human antibody. Preferably, the antibody is substantially fully humanised.

Preferred features of the second aspect of the present invention are as defined in relation to the first aspect of the present invention.

Advantageously, the use of optimised and/or humanised antibodies reduces or may even substantially avoid problems associated with immunogenicity, for example, problems associated with the immune system of a patient recognising an administered antibody as 'non-self and mounting a neutralising response.

Methods of determining the sequence homology of two protein sequences will be known to a person skilled in the art. For example, computational methods may be used.

The optimised and/or humanised antibody and derivatives thereof of the second aspect of the present invention may be produced by any suitable method. Suitable methods will be known to a person skilled in the art. For example, the optimised and/or humanised antibody and derivatives thereof may be produced using genetic engineering technology, chimaeric technologies, CDR grafting or veneering.

In certain embodiments, when the optimised and/or humanised antibody and derivatives thereof are produced using genetic engineering technology, a polynucleotide encoding the antibody may be isolated and cloned into an expression vector to obtain a recombinant plasmid, transforming a host organism with the obtained recombinant plasmids, culturing the transformants and causing expression of the polynucleotide encoding the antibody. When more than one polynucleotide is used, each polynucleotide may be cloned into the same or different expression vectors. Any suitable host organism may be transformed with the obtained recombinant plasmids. For example, the host organism may be prokaryotic, such as E.coli, bacilli including Bacillus subtilis and enterobacteriaceae including Salmonella typhimurium, or eukaryotic, such as yeast including Saccharamyces cerevisiae. It will be appreciated by a person skilled in the art that the step of cloning into an expression vector to produce a recombinant plasmid may be performed using standard methods in the same or different organism to the host organism. Preferably, the step of cloning into an expression vector to produce a recombinant plasmid may be performed using standard methods in E.coli.

The optimised and/or humanised antibody of the second aspect of the present invention may bind to any suitable antigen. In certain embodiments, the optimised and/or humanised antibody of the second aspect of the present invention may bind to monomeric C- reactive protein (mCRP). Preferably, the optimised and/or humanised antibody binds to the cholesterol binding region (CBR) and optionally to the C-terminal octapeptide region of monomeric C-reactive protein (mCRP). Preferred features of the mCRP are as defined above in relation to the first aspect of the present invention. Preferably, the optimised and/or humanised antibody of the second aspect of the present invention may bind to human mCRP, more preferably to the cholesterol binding region (CBR) and optionally to the C- terminal octapeptide region of human mCRP.

In certain embodiments, the antibody or antibody mixture may be used in the prevention, management or treatment of a neurodegenerative condition. Preferably, the antibody of any of the first, second and third aspects of the present invention may be used in a composition for use the in the prevention or treatment of neurological degradation or dementia.

The compositions and/or antibody or antibody mixtures may further comprise a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable', and like terms as used herein, means that the carrier is inert and does not cause harm to a human or other animal, such as a mammal, to which the composition is administered. Suitable pharmaceutically acceptable carriers will be known to a person skilled in the art and are described in, for example, Remington's Pharmaceutical Sciences, 16^th edition, (1982), Mack Publishing Co. For example, the pharmaceutically acceptable carrier may be water, a mixture of water and an alcohol, saline (i.e. sodium chloride solution), Ringer's solution, dextrose solution or buffered media. Preferably, the pharmaceutically acceptable carrier may be buffered with suitable buffer(s) to a pH of about 5 to 8, more preferably to a pH of about 6 to 8, most preferably to a pH of about 7. Preferably, the pharmaceutically acceptable carrier may be sterilised.

The antibody according to any of the fourth, fifth, sixth or seventh aspects of the present invention may be present in the pharmaceutically acceptable carrier in any suitable form. For example, the antibody may be present in the form of a solution, an emulsion or a dispersion in the pharmaceutically acceptable carrier. In certain embodiments, the antibody may be lyophilised (freeze dried), for example for storage and/or transport, and reconstituted in a suitable pharmaceutically acceptable carrier prior to use. Suitable pharmaceutically acceptable carriers are as defined above. Suitable methods of lyophilisation will be known to a person skilled in the art.

Other ingredients may also be used such as antimicrobials, antioxidants, chelating agents, inert gases, other therapeutic agents or medicaments or a combination thereof.

The antibody or antibody mixtures may be present in the composition according to any of the fourth, fifth, sixth or seventh aspects of the present invention at any suitable concentration. Preferably, the antibody may be present at a concentration of about 0.1 nanograms (ng) to 100 micrograms (mg), more preferably about 1 ng to 50 mg, most preferably about 10 mg to 50 mg.

The compositions and antibody or antibody may be for use in the (and/or a method of) prevention, management, mitigation or treatment of a neurodegenerative condition. 'Prevention, management, mitigation or treatment', and like terms as used herein, means that the composition may be administered before the onset of a neurodegenerative condition in order to prevent said onset or may be administered after the onset of a neurodegenerative condition in order to treat said neurodegenerative condition. The invention may be used in the prevention or treatment of any suitable neurodegenerative condition. For example, the composition may be used in the prevention or treatment of vascular dementia or Alzheimer's disease. In certain embodiments, the compositions may be used in the prevention or treatment of vascular dementia. In other embodiments, the composition may be used in the prevention or treatment of Alzheimer's disease. In yet a further embodiments, the composition may be used in the prevention or treatment of neurological and neurovascular conditions involving inflammation and leading to neurodegeneration. The neurological and neurovascular conditions involving inflammation may originate from brain ischemia, such as hemispheric ischemia, lacunar ischemia or stroke, brain injury, such as traumatic brain injury (TBI), hereditary vasculopathy, such as, for example, cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and combinations thereof. In an embodiment of the invention, the composition may be used in the prevention or treatment of vascular dementia, such as Alzheimer's disease, originating from a stroke. It will be known to a person skilled in the art that a stroke is typically caused by an ischemic event in the brain.

The composition may be administered by any suitable method. For example, the composition may be administered by injection, such as by intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal injection, or by continuous or non-continuous infusion, such as via a catheter. In certain embodiments, the administration may comprise a single dose. In other embodiments, the administration comprises multiple doses.

In accordance with a further aspect, there is provided a composition for use in the (or in a method of) treatment, prevention or management of a neurodegenerative condition comprising an antibody or peptide analogue thereof which binds to monomeric C-reactive protein (mCRP), wherein the composition is administered in a therapeutically effective amount:

(a) after an ischaemic event;

(b) after a thrombotic or embolic vessel occlusion; (c) after a neurovascular or traumatic event or acute or chronic hypoperfusion; or

(d) prophylactically to individuals at high risk of ischaemic events or having a history of ischaemic events.

The antibody or peptide analogue thereof may modulate the activity of the cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP.

For the avoidance of doubt, by 'prophylactically', or like terms as used herein, is meant that the composition is administered in order to substantially prevent or slow down the onset of a neurodegenerative condition brought about by elevated levels of mCRP due to the occurrence of an ischaemic event. The composition may be administered once or may be administered at a suitable number of time points over a suitable period of time. For example, the composition may be administered once or may be administered according to any suitable dosage regime. It will be appreciated by a person skilled in the art that the dosage regime may typically depend upon factors such as, for example, the age, sex and condition of the patient.

Preferably, the composition is administered after an ischaemic event, a thrombotic or embolic vessel occlusion event, a neurovascular or traumatic event or chronic hyperfusion event.

Preferably, the composition is administered up to about 3 months after an event. More preferably, the composition is administered up to about 1 month after an event. Even more preferably, the composition is administered up to about 1 week after an event. Yet more preferably, the composition is administered up to about 72 hours after an event. Even more preferably, the composition is administered up to about 48 hours after an event. Yet even more preferably, the composition is administered up to about 24 hours after an event. Most preferably, the composition is administered immediately after an event. It will be apparent to the skilled addressee that if multiple doses are required, then the composition may be administered in two or more doses over the range of 3 months to immediately after an event, over the range of 1 month to immediately after an event to be administered, over the range of 1 week to immediately after an event, over the range of 3 months to immediately after an event, over the range of 72 hours to immediately after an event, over the range of 48 hours to immediately after an event, over the range of 24 hours to immediately after an event. The two or more doses may comprise multiple doses administered at equally spaced time intervals through these ranges or as doses at both end points of the ranges.

'High risk of ischaemic events', and like terms as used herein, means an individual is at a higher risk than normal of ischemia, i.e. a restriction in blood supply to tissues. For example, the ischaemic event may be caused by vasoconstriction, thrombosis or embolism, trauma or asphyxia. Individuals at a high risk of ischemia may have a pre-existing medical condition such as, for example, heart disease, hypertension, diabetes, hyperuricemia or high cholesterol.

The compositions of the invention may be administered in a number of ways, however it is preferred that such methods enable the composition to traverse the blood brain barrier.

In accordance with a further aspect, there is provided a kit of parts comprising the composition or the antibody or antibody mixture as herein above described, a receptacle in which said composition or antibody or antibody mixture is provided and equipment required for the administration of said composition or antibody or antibody mixture.

The receptacle in which said compound or antibody or antibody mixture is provided may be in any suitable receptacle. For example, the receptacle in which said antibody or peptide derivative is provided thereof may be a vial, such as a glass or plastic vial. Preferably, the receptacle in which said antibody or peptide derivative thereof is provided may be a glass vial.

The equipment required for the administration may be any suitable piece of equipment. For example, the optional further equipment required for the administration such as an intraarterial delivery device, a needle, a syringe or a combination thereof.

The kit of parts may optionally further contain instructions for administering the antibody or peptide analogue thereof.

Combinations of the features of the invention are described in the following paragraphs: Paragraph 1. A composition for use in a method of treatment, prevention or management of neurological degeneration or dementia, comprising an inhibitor or antagonist of monomeric C-reactive protein (mCRP).

Paragraph 2. The composition of paragraph 1 , wherein the inhibitor or antagonist modulates the activity of the cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP.

Paragraph 3. The composition of paragraph 1 , wherein the inhibitor or antagonist binds to amino acid residues of mCRP amino acid residues of about 35 to about 47 and/or about 199 to about 206.

Paragraph 4. The composition of paragraph 1 , wherein the inhibitor or antagonist binds to at least part of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% homology thereof.

Paragraph 5. The composition of paragraph 1 , wherein the inhibitor or antagonist comprising a peptidic moiety according to a Formula 1 or salt, derivative, prodrug or mimetic thereof:

Formula 1 : [X1-X2-X3-X4-X5-X6]

wherein X1 may be present or absent, when X1 is present, X1 comprises V;

wherein X2 may be present or absent, when X2 is present, X2 comprises C;

wherein X3 comprises L;

wherein X4 comprises H, R, G, W or Y;

wherein X5 comprises up to 170 amino acids; and

wherein X6 may be present or absent, when X6 is present, X6 comprises FTKPQLWP. Paragraph 6. The composition of paragraph 5, wherein X5 comprises C at position 59 if X2 is present.

Paragraph 7. The composition of paragraph 5, wherein X1 to X5 comprises VCLHFYTELSSTR. Paragraph 8. The composition of paragraph 5, wherein the composition comprises amino acid residues or amino acid analogues.

Paragraph 9. The composition of paragraph 5, wherein the composition comprises a peptide or peptide mimetic molecule.

Paragraph 10. The composition of paragraph 1 , wherein the inhibitor or antagonist comprises an antibody or antibody mixture.

Paragraph 11. The composition of paragraph 1 , wherein the neurological degeneration or dementia is related to or due to inflammation.

Paragraph 12. The composition of paragraph 1 1 , wherein the inflammation is the result of neurovascular or traumatic event or to acute or chronic hypoperfusion.

Paragraph 13. The composition of paragraph 1 , wherein the neurological degeneration or dementia is related to or due to thrombotic or embolic vessel occlusion. Paragraph 14. The composition of paragraph 1 , wherein the neurological degeneration or dementia is related to or due to an ischaemic event.

Paragraph 15. An antibody or antibody mixture, for use in a method of treatment, prevention or management of neurological degeneration or dementia, wherein the antibody or antibody mixture is capable of binding to the cholesterol binding region (CBR) and optionally to the C-terminal octapeptide region of monomeric C-reactive protein (mCRP).

Paragraph 16. The antibody or antibody mixture of paragraph 15, wherein the antibody binds to mCRP between or at amino acid positions of about 35 to about 47 and optionally within about 199 to about 206.

Paragraph 17. The antibody or antibody mixture of paragraph 15, wherein the antibody binds to at least part of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and optionally FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% homology thereof.

Paragraph 18. A composition for use in a method of treatment, prevention or management of a neurodegenerative condition comprising an antibody or peptide analogue thereof which binds to monomeric C-reactive protein (mCRP), wherein the composition is administered in a therapeutically effective amount:

(a) after an ischaemic event;

(b) after a thrombotic or embolic vessel occlusion;

(c) after a neurovascular or traumatic event or acute or chronic hypoperfusion; or

Paragraph 19. The composition of paragraph 18, wherein the composition is administered up to about 72 or 48 hours after an ischaemic event.

Paragraph 20. The composition of paragraph 18, wherein the composition is administered during or immediately after an ischaemic event.

Paragraph 21. The composition of paragraph 18, wherein the composition is administered prophylactically to individuals having elevated levels of mCRP.

Paragraph 22. The composition of paragraph 18, wherein the composition is administered intra-arterially.

Paragraph 23. A kit of parts comprising an inhibitor or antagonist of monomeric C- reactive protein (mCRP) or an antibody or antibody mixture wherein the antibody or antibody mixture is capable of binding to the cholesterol binding region (CBR) and optionally to the C-terminal octapeptide region of monomeric C-reactive protein (mCRP) and equipment required for the administration of said composition or antibody or antibody mixture.

Paragraph 20. The kit of paragraph 23, wherein the equipment comprises intraarterial delivery device.

Detailed Description of the Invention

The present invention will now be further described with reference to the following non- limiting examples and figures in which:

Figure 1 : Kinexus Western phospho-microarray analysis and Western blotting of mCRP-induced signalling in BAEC. (A) shows quantitative Kinexus phospho-protein screening array carried out on BAEC after exposure to mCRP (8 minutes) demonstrated up- regulation of several potentially important proteins that may be implicated in AD pathology including Tau (2.3 fold) Focal adhesion kinase and IRS-1 (3.4 fold). IRS-1 was investigated in more detail in the in vitro studies. Figure 1 B shows by Western blotting in the same samples, that mCRP induced approximately a fourfold increase in p-IRS expression compared with control untreated cells (bar chart). P-Tau was also increased by approximately 5-fold (C) and in addition, it was shown that the cellular content of Αβ1-42 increased 3-fold over 24h whilst PEN-2 also increased (2-fold; 8 minutes, D). These experiments were carried out at least twice and a representative example is shown.

Figure 2: Effects of siRNA knock-down of IRS-1 on BAEC angiogenesis and cell signalling: BAEC were subjected to siRNA knock-down of IRS-1 as described in the Materials and Methods section. After 48h treatment, approximately an 85% reduction in IRS-1 gene expression was noted (A). In contrast, NC siRNA had no effect on IRS-1 gene expression. Knock-down was tested for each experiment and found to be similar and the figure shows a representative example. (B), a reduction (50%) in mCRP-induced tube-like-structure formation was seen in siRNA-treated cells. The bar chart shows significant reduction in tube formation in the presence of IRS-1 siRNA (**p<0.01 ; *p<0.05). Pre-incubation with the characterised antibody specific for mCRP (4h; ^g/ml) was able to inhibit both p-ERK1/2 and p-IRS-1 expression in BAEC (C), and also, significantly, tube-like-structure formation (D; *p<0.05). Note pCRP was not tested in these assays as we have previously shown that it has no pro-angiogenic activity. These experiments were carried out three times and where statistical analysis was performed, results represent the mean ± S.D of these experiments.

Figure 3: Characterization of mCRP-induced vascular activation: A, BAEC spheroids were generated to examine the effect of mCRP on sprout structure and formation in a 3- dimensional system. In normal culture conditions, sprouting was slower, sprouts had a thicker appearance and cell-cell junctions were maintained (left panel). In the presence of mCRP, sprouts formed more quickly, were notably thinner in appearance and the intercellular gaps between cells was notably larger (right panel). B, Dorsal matrigel implants containing mCRP (1C^g/ml; 72h) produced strong and visible haemorrhagic angiogenesis (iv; arrows) compared with a typical, normal looking vascular response seen in the presence of VEGF (ii; 25ng/ml), whilst pCRP (1 C^g/ml) produced very little angiogenic response (p<0.05 increase in the presence of mCRP and VEGF compared with control implants) (iii). In C, the graph shows a significant increase in monolayer permeability in the presence of mCRP (1 C^g/ml; 8h) using a Millipore-based filter assay, similar to that produced by 10% DMSO (p<0.01 increase in FITC dextran penetrating the monolayer in the presence of either mCRP or the positive control DMSO), and lighter regions in the images shown indicate areas of increased permeability. (D), Expression of adhesion molecules was examined in BAEC treated with mCRP (10μg/ml; 24h). NCAM expression was increased by approximately 2.8 fold whilst VCAM, ICAM and integrins were not affected (data not shown), β-tubulin was used as the house keeping control (gel and bar chart shown). These experiments were repeated at least twice and a representative example is shown.

Figure 4: Behavioural changes observed following mouse-hippocampal injection of mCRP. Stereotactic injection of mCRP (50μg) directly into the CA1 hippocampal region of mice were examined 3-4 weeks after operation. A significant reduction in balance and grip was seen in non-transgenic animals in the presence of mCRP (wire hang test; A), whilst head dipping latency in the Boissier's hole-board test was significantly increased in 3xTg mice and further increased when mCRP was present (B). In the same test, the latency to entry into 4 holes was significantly increased in both mCRP-treated non-transgenic and 3xTg mice (C). Statistical analysis was done using two-way ANOVA (*P<0.05).

Figure 5: Cognitive effects of mCRP on mice following hippocampal injection. (A-C), shows results for novel object recognition. Whilst no effects were seen at time zero, a trend was seen after 2h and after 24h, visual discrimination ratio was significantly decreased in the presence of mCRP in both non-transgenic and 3xTg mice (C). Similarly, in the water maze test (D-F), mCRP-treated mice showed a significant increase in distance covered (D) and also reduction of % time in the target quadrant (E-F). Statistical analysis was done using two- way ANOVA (*P<0.05). Figure 6: IHC of mouse brain tissue sections following mCRP hippocampal injection. Example staining results from histological and ICH analysis of mouse brain tissue sections (5μ). A(i-iii), shows mCRP-positive ventricles (i), neurons close to the injection site (ii) and positive neuronal staining around cortical ventricular tracts (iii). (iv) There are numerous positively (peri-nuclear) stained irregular hypertrophic looking cortical neurons. In (v), CA1 hippocampal neurons show strong mCRP-positivity whilst in (vi), distant staining was observed in neurons of the hypothalamic region. In (vii)-(viii), cortical microvessels are clearly stained for mCRP. (ix) shows non-transgenic control brain hippocampal neurons negative for mCRP staining and (x) shows cortical neurons also negatively stained for mCRP.

(B) P-Tau positivity was increased in sections of cortical neurons, (i) shows an example of negative staining in hippocampal neurons of a control mouse whilst (ii) shows p-tau staining in cortical neurons from a 3xTg mouse and (iii) an equivalent section from an mCRP- injected animal, (iv) shows notable hippocampal (peri-nuclear) staining whilst (v) shows positive axonal cortical neuronal staining (v).

Αβ staining is shown in C (i) shows negative staining in a normal non-injected mouse,

(ii) and (iii) show plaque-like element staining and neuronal axons with peri-nuclear staining respectively, whilst (iv) and (v) show sections of 3xTg mice demonstrating a similar staining pattern.

D shows p-IRS-1 and mCRP staining in cortical neurons and plaques (i-ii; serial sections) and matching areas of serial sections showing co-localization in ventricular tracts local to the injection site (iii-iv), CA1 hippocampal neurons (v-vi). vii-viii, shows the presence of p-IRS-1-labelled plaque-like mCRP-positive structures in the same region as mCRP positive areas, (ix) shows non-transgenic mouse cortical region negatively stained for p-IRS- 1.

E shows double immunofluorescent micrographs demonstrating Ca1 neuronal co- localization of mCRP (TRITC) and p-Tau (i-iii; plus insert), and in cortical microvessels (iv; CD31=FITC). Areas of direct co-localization appeared yellow. Due to antibody binding restrictions, active angiogenic vessels were labelled with CD105 in serial sections and compared with mCRP staining, (v)-(vi) show mCRP and CD105 staining in the same vessels of serial sections respectively. No positively stained microvessels were observed in the normal non-transgenic mouse cortex (data not shown). Magnification bars 2.5mm= x 400.

Figure 7: Western blotting showing mCRP-neuronal protein phosphorylation. Rat cortical neurons cultured in basal medium showed approximately an 8.5 fold increase in p- Tau expression and a 5-fold increase in p-ERK1/2 by Western blotting after 8 minutes treatment with mCRP (i-ii respectively; "^g/ml). In addition, (ii) mCRP induced increased phosphorylation of p-IRS-1 (5 fold), p-Akt (2.5 fold) and p-APP (2 fold), (iii) shows that preincubation with our anti-mCRP blocking antibody was able to inhibit mCRP signalling through p-ERK1/2 and p-Tau. These experiments were carried out at least twice and a representative example is shown, (iv) Kinexus phospho-protein Western array carried out on control neurons versus mCRP-treated cells (8 minutes; "^g/ml) revealed further proteins that could be involved in neuronal-mCRP signalling including focal adhesion kinase (2.2 fold increase) and p-53 (1.7 fold increase). Magnification bars 2.5mm= x 400.

Figure 8: Tau fibrilization assay. In vitro assay showing Tau 244-372 aggregation induced by mCRP (10μg/ml; 24h) (c), with a similar profile to that produced by the positive control arachidonic acid (150μΜ). (a) shows the control Tau incubated with all other buffer component's minus CRP or arachidonic acid (b). These experiments were carried out at least twice and a representative example is shown. Magnification bars 10mm = 1 μΜ.

Figure 9: Immunohistochemistry showing localization of mCRP i-ii) in the regions containing β-amyloid-positive plaques (iii) of patients with AD following stroke (Ai-Aiii; arrows; patient 6). Expression can also be seen at the higher magnification in affected neurons (x 200). Figure 9B shows strong mCRP staining in microvessels and early stage neuritic plaques from patient 6 who had suffered previous ischaemic stroke (Bi-Bii; arrows; x 40) and Biii shows a cortical region near to the infarcted zone that is strongly positive for mCRP in microvessels and plaques. mCRP appeared to stain NFTs in these regions (Ci-ii) whilst a strong co-localization of mCRP (TRITC) with Αβ (FITC) (D) and CD105 (DAB)-suggesting angiogenesis (E). Magnification bars 2.5mm= x 400. Figure 10: Patient 8, (i) serial sections of stroke-affected cortex showing an almost identical pattern of staining with mCRP antibody (top) and p-Tau (bottom). Magnification x 200. (ii) Patient 6 low magnification of mCRP staining (DAB) showing the micro-infarct localization (arrow) and curved lines demonstrating as we move further away from the core, the mCRP staining becomes weaker and weaker. Magnification bars 2.5mm= x 400.

Figure 1 1 : A schematic diagram highlighting probable novel and key signalling intermediates associated with mCRP-cell interactions which could contribute to development of vascular dementia/AD.

Figure 12: Sequence Listings. SEQ ID No. 1 is the amino acid sequence for mCRP; SEQ ID No. 2 is the amino acid sequence of the CBS region of human mCRP; and SEQ ID No. 3 is the amino acid sequence of the the C-terminal octapeptide region of human mCRP. Materials and Methods:

Monomeric and pentameric CRP source

Recombinant forms of both mCRP and pCRP (0.5 mg/mL in 25 mM NaPBS, pH 7.4) was produced. The mCRP solution contained an endotoxin concentration lower than 0.125 EU/mL and all cell culture medium was endotoxin-free.

Antibodies to mCRP and pCRP

Mouse monoclonal antibodies to human pCRP (N2C10), and the octapeptide of the mCRP sub-unit (M8C10) were obtained and fully characterized (as described in Slevin M., et al. Modified C-reactive protein is expressed by stroke neovessels and is a potent activator of angiogenesis in vitro. Brain Pathol, 20, 151-165. (2010)).

Cell culture

Bovine aortic endothelial cells (BAEC) were isolated and seeded in 75-cm² flasks pre- coated with 0.1 % gelatine (Sigma, UK) and cultured in Dulbecco's Modified Eagle Medium (Lonza, UK) supplemented with 20% foetal bovine serum (FBS, Cambrex, UK), 2 mM glutamine and 1 % antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin). BAEC were placed at 37°C in a saturated air humidity/5% C0₂-incubator. Every 2-3 days, the cells were detached by enzymatic digestion with 0.05% trypsin / 0.02% EDTA and split at a ratio of 1 :2 or 1 :3. At confluence, EC were identified by their typical cobblestone morphology and "hill and valley" configuration, respectively. The cells were used throughout the study between passages 4 and 9. Rat cortical neurons (RCN) were obtained from Life Technologies (UK) and cultured directly into T-25 flasks. These were used at the same cell concentration and under the same conditions as BAEC, and according to the manufacturer's instructions.

Endothelial tube formation assay

BAEC (1.5x10⁶ cells/ ml) were mixed in equal volume with growth factor-reduced Matrigel™ (10 mg/ml) with or without 5 μg/ml mCRP, and a spot of the mixture was poured into the centre of each well in a 48-well plates (Nunc). After polymerization of the gel for 1 h at 37°C, each spot of cells embedded in Matrigel™ was bathed in 500 μΙ of complete medium. After 24 h incubation, some cells migrated and aligned to form tubes (defined by the enclosure of circumscribed areas), a parameter of quantification. For the counting of enclosed areas, the cells were fixed with 4% PFA for 15 min and counts made in five fields by microscopy using the x 20 objective. Using the same experimental procedure described above, BAEC were mixed with anti-mCRP antibodies (^g/ml) at the same time as matrigel and mCRP ^g/ml) to assess if the antibody was capable of blocking mCRP-induced angiogenesis. Experiments were repeated three times and results are shown as mean ±S.D. (*p<0.05).

Spheroid sprouting assay for junctional analysis by T.E.M.

Cells were harvested from sub-confluent monolayer cultures by trypsinization and

6x10⁵ cells were suspended in DMEM 10% FBS and 0.25% (w/v) carboxymethylcellulose (Sigma, UK). Cells were then seeded into non-adherent round-bottom 96-well plates to assemble into a single spheroid within 24 h at 37 °C, 5% C0₂. Basic fibroblast growth factor (human recombinant FGF-2, (BD Bioscience, UK)) was used as positive control and added at a final concentration of 25 ng/ml. After 24h, gels were photographed and sprouting architecture was assessed microscopically. Experiments were performed in triplicates.

Endothelial cell permeability assay BAEC were cultured to confluence on Transwell collagen coated permeable (pored) supports (Millipore) and medium replaced with SPM (24h). mCRP (1 C^g/ml) or DMSO (^g/ml; positive control) was added and cells incubated at 37°C for a further 8h. Next, the medium was refreshed and replaced with 500μΙ of basal medium and a solution of FITC- dextran (150μΙ; 1 :20 added to each well and incubation continued at room temperature for a further 4h). The quantity of FITC-dextran passing through the pores of the insert into the collecting plate was proportional to the permeability of the monolayer. 10ΟμΙ of the collecting medium was read on a fluorescent plate reader with filters at 485 and 535nm excitation and emission according to the manufacturer's instructions. Photomicrographs of the monolayers stained with Millipore cell stain (10 minutes) were also obtained. Each test was performed in triplicate and the experiment performed twice, a representative example is shown and p<0.01 is indicated by *).

Mouse matrigel implant

In this study (n=3 per group), aliquots of growth factor reduced matrigel (400μΙ) were injected into C57BL/6 mice subcutaneously at the dorsal surface, +/- addition of VEGF (positive control 10μg/ml) or pCRP/mCRP (10μg/ml). After 5 days, the animals were euthanized and plugs dissected and photographed in order to identify evidence of vascularization and neo-circulation. Simple statistical analysis (mean ±S.D. was used to count the numbers of vessels associated with each implant. Detailed histological analysis of the plugs was not carried out.

Short-Interfering RNA (siRNA) targeting IRS-1

Short-interfering RNA duplexes targeting bovine IRS1 (IRS1 siRNA) and negative control (NC siRNA) were selected and chemically synthesised by GenePharma (Shanghai, China). Their sequences were:

5' GCGGUAGUGGCAAACUCUUTT 3' (sense)

5' AAGAG U U UGCCAC U ACCGCTT 3' (anti-sense)

To examine the transfection efficiency of Lipofectamine2000 (Invitrogen), NC-FAM (negative control-fluorescein amino-modified oligonucleotides) siRNA was transfected into BAEC previously seeded on glass coverslips, and the following day the cells were washed with PBS, fixed with 4% PFA and visualised with a Zeiss fluorescent microscope. For mRNA 'knock-down', the reverse transfection method was applied (as described by Mizoroki T., et al. Aluminium induces tau aggregation in vitro but not in vivo. J. Alzheimers Dis. 11, 419-427. (2007)). Briefly, the siRNA duplexes were transfected into 70-80% confluent BAEC cultured in a 24-well plate at a final concentration of 50 nM. The cells were incubated at 37°C for 2 hours before the addition of 10% FBS with 100 U/ml penicillin and 100 μg/ml streptomycin then cultured for a further 24 h for RT-PCR, tube-formation analysis. Experiments were repeated three times and results shown as the mean ±S.D (*p<0.05).

RNA extraction and RT-PCR

Extraction of total RNA from BAEC was performed and complementary DNA (cDNA) was produced from total RNA extracts in two stages as described by the standard protocols and subsequently used to monitor bovine IRS1 mRNA expression. The gene-specific primer pairs used for PCR were optimized for cycle number (IRS1 = 35 cycles) and t_m (58°C). Primer pairs (Invitrogen) were selected by software Primer3 and the sequences are shown above. PCR products were analysed by 1.5% agarose gel electrophoresis. Ribosomal protein S14 expression was used as an internal standard.

Kinexus phospho-protein array analysis

BAEC or RCN were seeded in 6-well plates at a concentration of 3 x 10⁵ cells/2 ml in complete medium. After 48 h incubation, the medium was replaced with serum-free medium for a further 24 h incubation then 5 μg/ml mCRP was added to the cells and allowed to incubate for 8 min at 37°C. To screen the modulation of the signalling protein expression downstream of the mCRP receptor, a phospho-protein micro-array analysis of 500 phospho- site proteins, Kinex™ Antibody Microarray (KAM)-1.2 was performed by Kinexus Bioinformatics (Vancouver, Canada). Proteins from mCRP-stimulated and un-stimulated cells were extracted according to Kinetworks instructions. Briefly, cells were washed twice with cold PBS. Protein was extracted using a buffer (pH 7.2) containing 20 mM MOPS, 2mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 60 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 μΜ pepstatin A, 10 μΜ leupeptin, 1 % Triton X-100 and 1 mM dithiothreitol on ice for cell lysis. Cell extracts were then collected and protein concentrations were measured by the Bradford protein assay using a plate reader and according to the manufacturer's instructions (Bio-rad, Munchen, Germany). For each sample, protein (100μg in 50μΙ) was transferred to a fresh 1.5 ml Eppendorf screw cap and this was sent to Kinexus for analysis. Western blot Analysis

BAEC/RCN were seeded in complete medium in a 24-well plate at a cell concentration of 10⁵/ml/well. After 48 h incubation, the medium was renewed with SPM and cells incubated for a further 24 h. Next, 5 μg/ml mCRP was added and the cells incubated for 8 min at 37°C. In addition, BAEC cultured under the same conditions were exposed to mCRP antibody (^g/ml) immediately prior to addition of mCRP in some experiments in order to identify any potential blocking effects on cell signalling. After rapid washing in cold PBS, cells were lysed with 120 μΙ/well of ice-cold radioimmunoprecipitation (RIPA) buffer (pH 7.5) containing 25 mM Tris-HCI, 150 mM NaCI, 0.5% sodium deoxycholate, 0.5% SDS, 1 mM EDTA, 1 mM sodium orthovanadate (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 % Triton X100 and 1 μΜ leupeptin. The protein concentration of cell lysates was determined using the Bradford protein assay (Bio-rad, Munchen, Germany) and equal quantities of proteins (15 μg) were mixed with 2 X Laemmli sample buffer, boiled in a water bath for 15 min then centrifuged. Samples were separated along with pre-stained molecular weight markers (32,000 - 200,000 Da) by 12% SDS-PAGE. Proteins were electroblotted (Hoefer, Bucks, UK) onto nitrocellulose filters (1 h) and the filters were blocked for 1 h at room temperature in TBS-Tween (pH 7.4) containing 1 % bovine serum albumin (BSA). Filters were stained with the following primary antibodies diluted in the blocking buffer, overnight at 4°C on a rotating shaker: rabbit monoclonal antibodies to phospho-IRS1 (Y1 179), rabbit polyclonal antibodies to γ-secretase subunit presenilin enhancer protein 2 (1 :1000) from Acris Antibodies (San Diego, CA, USA), p-Tau (S404), NCAM, (1 :1000), p-APP (Y757), PEN-2, p-ERK1/2 and β- amyloid (1-42; Abeam, UK; in this case, cells were cultured with mCRP for 24h) and mouse monoclonal antibodies to β-tubulin (1 : 1000) from Santa Cruz Biotechnology. After washing (5 x 10 min in TBS-Tween at room temperature), filters were stained with either goat anti-rabbit or rabbit anti-mouse horse-radish peroxidase-conjugated secondary antibodies diluted in TBS-Tween containing 5% de-fatted milk (1 :1000, 1 h, room temperature) with continuous mixing. After a further five washes in TBS-Tween, proteins were visualized using ECL chemi- luminescent detection (Geneflow) and semi-quantitatively identified fold differences compared with house-keeping controls determined using Image-J software. All experiments were repeated at least twice and a representative example is shown.

NFT- Aggregation of Tau (human protein, Sigma)

The aggregation of Tau₂44-3₇2 (8 μΜ) was induced by 150 μΜ of arachidonic acid

(Sigma) in buffer containing 10 mM Hepes (pH 7.6), 100 mM NaCI, 5 mM dithiothreitol for 24 h incubation at room temperature without stirring. To detect a direct effect of mCRP on Tau aggregation, Tau was incubated with mCRP (10 μg/ml) in the same buffer (minus arachidonic acid) for 24 h at room temperature. The protein samples were fixed with 2% glutaraldehyde (Sigma), ultracentrifuged at 100,000g for 30 minutes then spread on glass coverslips and allowed to dry in air. The samples were then sputter coated with gold and examined in a Jeol JSM-5600LV scanning electron microscope at an accelerating voltage of 12 kV and 10,000 x magnification. Experiments were carried out at least twice and a representative example is shown.

Animals

Twenty four male 3xTg-AD mice and twenty four non-transgenic (NTg) mice were used in this study. The 3xTg-AD mouse model (primarily used here as a positive AD-like control) was genetically engineered at the University of California, Irvine to express the familial AD mutations PS1/M146V, APPswe and tauP301 L (as described in Revilla S., et al. Physical exercise improves synaptic dysfunction and recovers the loss of survival factors in 3xTg-AD mouse brain. Neuropharmacology. 81, 55-63. (2014)). The NTg mice had the same genetic background hybrid 129 x C57BL6 as 3xTg-AD. Mice were bred from the Spanish colony established in the Medical Psychology Unit, Autonomous University of Barcelona. Genotypes were confirmed by polymerase chain reaction (PCR) analysis of DNA obtained from ear punches. Animals were individually housed in Macrolon cages (Techniplast, Buguggiatta, Italy) with free access to food and water and maintained in a temperature controlled room (22 ± 2 °C) with 12 hours light/12 hours dark cycle.

Hippocampal injection of mCRP mCRP, was delivered into the CA1 region of the mouse hippocampus by stereotactic surgery procedures. Four-month old mice (n=8 per group) were anesthetized with 10 mg/kg xylacine (Rompun 2%, Bayer, Leverkusen, Germany) i.p. and 80 mg/kg ketamine (Ketolar 50 mg/ml, Pfizer, Alcobendas, Madrid, Spain) i.p. and placed in a stereotactic apparatus (David

Kopf Instruments, Tujunga, CA). Bilateral infusions of either an experimental agent solution or artificial CSF (NaCI 148 mmol/l, KCI 3 mmol/l, CaCI2 1 mmol/l, MgCI2 0.8 mmol/l, Na2HP04 0.8 mmol/l, NaH2P04 0.2 mmol/l) were performed into the CA1 area of the hippocampus. Solutions of mCRP contained a final content of 50μg. Injections were performed at a rate of Ι μΙ/min at coordinates relative to Bregma of -2.0 mm A/P, ±1.2 mm

M/L, -1.5 mm V/D. One microliter of the testing solutions was delivered to the application point with a 25-gauge stainless steel cannula (Small Parts Inc., Miami, FL) connected to a Hamilton syringe through a Teflon tube. The syringe was attached to a micro-infusion pump (Bioanalytical systems Inc., West Lafayette, IN). The cannula was left in position for 5 min after delivery to prevent the solution from surging back. One animal died immediately after surgery; otherwise, eight mice formed the experimental groups. Behavioral testing

Animals were tested for changes of non-cognitive and cognitive behavior at two-three weeks after hippocampal injections. A battery of tests was applied on fourteen daily consecutive sessions.

Sensorimotor responses: Visual reflex and posterior legs extension reflex were measured by holding the animal by its tail and slowly lowering it towards a black surface. Motor coordination and equilibrium were assessed by the distance covered and the latency to fall off a horizontal wooden rod and a metal wire rod, as described in Garcia-Mesa Y., et al. J Alzheimers Dis. 24,421-54. (2011). Prehensility and motor coordination were measured as the distance covered on the wire hang test, which consisted of allowing the animal to cling from the middle of a horizontal wire (2 mm diameter x 40 cm length) with its forepaws for two trials of 5 s and a third 60 s trial.

Corner test: Neophobia to a new home-cage was assessed by introducing the animal into the center of a standard square cage (Macrolon, 35 x 35 x 25 cm) with fresh bedding and counting the number of corners visited and rearings during a period of 30 s. The latency of the first rearing was also recorded.

Open field test: Mice were placed in the center of the apparatus (home-made, wooden, white, 55 x 55 x 25 cm high) and observed for 5 min. Patterns of horizontal locomotor activity (distance covered and thigmotaxis) and vertical movement (rearings) were analyzed throughout the test. Initial freezing, self-grooming behavior and the number of urine spots and defecation boli were also recorded.

Dark and light box test: Anxiety-like behavior was measured in a dark-light box. The apparatus consisted of two compartments (black: 27 x 18 x 27 cm with a red light; white: 27 x 27 x 27 cm with white lighting intensity of 600 lux) connected by an opening (7 x 7 cm). The mice were introduced into the black compartment and observed for 5 min. The latency to enter the lit compartment, the time spent in the lit compartment and the number of rearings were recorded.

Boissier's four hole-board test: Exploratory behavior was measured as the number of head-dips and time spent head-dipping on each of the four holes (3 cm diameter) equally spaced in the floor of the hole-board (woodwork white box of 32 x 32 x 32 cm). The latencies of movement, first dipping and four hole dipping were recorded.

Tail suspension test: Mice were suspended by the tail to assess depression-like behavior. The mouse was hanged 30 cm above the surface. The tail was fixed with adhesive tape at 1 cm from its tip. The duration of immobility (defined as the absence of all movement except for those required for respiration) was scored during 6 min. Object recognition test: Animals were placed in the middle of a black maze with two arms angled 90°, each measuring 25 cm x 5 cm. The 20 cm high walls could be lifted off for easy cleaning. The lighting intensity was 30 lux. The objects to be discriminated were made of wood (5-6 cm high, brightly colored). After two previous days of habituation, the animals were submitted to a 10 min acquisition trial (first trial) during which the mouse was placed in the maze in the presence of two identical novel objects (Α+Α') placed at the end of each arm. A 10 min retention trial (second trial) occurred 2 h later, replacing object A' in the maze by object B. Another 10 min retention trial (third trial) occurred 24 h later, replacing object A in the maze by object C. The time that the animal explored the new object and the old object were recorded. In order to avoid object preference biases, the sequence of presentation of the different objects was counter-balanced in each experimental group. The maze and the objects were cleaned with 96% ethanol between different animals, to eliminate olfactory cues.

Morris water maze test: Animals were tested for spatial learning and memory in the Morris water maze (MWM), consisting of one day of cue learning and six days of place task learning for spatial reference memory, followed by one probe trial. To test the spatial learning acquisition, mice were trained to locate a hidden platform, 10 cm in diameter, located 20 cm from the wall and 0.5 cm below the water surface. This was placed in a circular pool 100 cm in diameter, 40 cm height, with 25°C opaque water, surrounded by black curtains. The animals learned to find the platform using distinctive landmarks as visual cues (four trial sessions of 60 s per day). On day 7, after one trial of place learning, the platform was removed and the mice performed a probe trial of 60 s to test the retention of learning. A computerized tracking system (SMART, Panlab S.A., Barcelona, Spain) allowed to measure the distance covered during the learning tasks, along with the time spent in each quadrant of the pool after the removal of the platform in the probe test. For statistical analysis, 2-way ANOVA was used and significance was defined as *p<0.05.

Histological analysis After completion of the behavioral tests, at 1 month after injection, mice were anesthetized as described above and transcardially perfused with 100 mM phosphate buffer (PB, pH 7.4) containing 0.1 mg/ml heparin (Mayne Pharma, Spain) followed by 4% paraformaldehyde in PB. Brains were removed and post-fixed overnight in cold paraformaldehyde, rinsed with cold PB and then dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin. Serial sections were cut throughout the brain at 5μηι, and I HC was carried out at 1 mm intervals throughout the brain-details described below in the section on immunohistochemistry (animals n=5 per group) in order to determine expression and localization of mCRP, p-Tau, p-IRS-1 and Αβ.

Anatomo-pathological study

Brain samples: 1) Ischaemic stroke with AD: Samples were obtained from the Institute of Neuropathology Brain Bank, University Hospital of Bellvitge, Catalonia and ethical approval for the work was granted. The tissue samples had been collected within 4 hours of death from the refrigerated bodies of 10 patients who died 2-29 days after stroke following middle cerebral artery occlusion (details are provided in Table 1 below).

Table 1 Patient details of Ischaemic stroke samples +/- AD from Belvitge Hospital

Table 1

Patients presented with dementia, confirmed in memory clinics and the diagnosis of probable AD was confirmed by anatomopathology. All the AD cases had a history of progressive dementia and were selected on the basis of a diagnosis according to CERAD of 'definite AD' and a Braak tangle stage of V-VI; according to NIA-Alzheimer's Association guidelines AD neuropathological change was considered a sufficient explanation for the dementia in all cases. Samples were dissected into infarcted (identified with 2, 3, 5- triphenyltetrazolium chloride), peri-infarcted and normal looking unaffected tissue as previously described. Peri-infarcted tissue showed structural integrity but was characterised by oedema, altered morphology of the neurons (some showing changes of apoptosis), and angiogenesis. Tissue from the contralateral hemisphere served as a control. Samples were dissected into 2 mm diameter pieces and either frozen in liquid nitrogen at -70°or fixed in 10% buffered saline prior to paraffin embedding.

2) Alzheimer's tissue (post mortem) from 20 patients and 10 negative controls was obtained from the Brain Bank in Bristol (UK). Tissue sections were obtained from blocks of the cerebral cortex, specifically, from the frontal lobe, parietal lobe and occipital lobe. Details are shown in Table 2 below.

Table 2 AD patient/specimen details from Bristol Brain Bank, UK

BB Braak PM

NO MRC ID DIAG 1 DIAG 2 DIAG 3 AGE SEX Stage DELAY APOE

1 19 BBN 8748 AD NO NO 91 F 6 78 3.4

217 BBN_8846 AD NO NO 84 F 6 96 4.4

221 BBN 8850 AD NO NO 84 F 5 41 4.4

270 BBN 8899 AD NO NO 92 M 5 45 2.4

317 BBN 8945 AD NO NO 91 F 4 12 4.4

341 BBN 8969 AD NO NO 95 M 3 48 3.3

434 BBN 9060 AD NO NO 87 M n/a 12 n/a

451 BBN 9076 AD NO NO 84 F 5 20 3.4

Argyrophilic

grain

456 BBN 9081 AD disease NO 92 M 2 84 n/a

465 BBN 9090 AD CAA NO 83 M 6 20 3.3

496 BBN_9120 AD NO NO 84 F n/a 24 2.3

530 BBN 9154 AD NO NO 81 M n/a 28 3.4

677 BBN 9255 AD NO NO 81 M n/a 16 n/a

691 BBN 9269 AD NO NO 83 M 4 99 3.4

697 BBN 9275 AD NO NO 87 M 6 36 4.4

731 BBN_9309 AD NO NO 88 F 4-5 88 3.3

745 BBN 9323 AD NO NO 84 F 6 20.5 2.3

760 BBN 9338 AD CAA SVD 95 M 4 27 3.4

768 BBN 9346 AD NO NO 88 F 6 64 3.3

821 BBN 4232 AD CVD NO 84 F 6 65.5 n/a 160 BBN 8789 CONT. NO NO 78 F 2 24 n/a

CONT.

323 BBN 8951 NO NO 96 F n/a n/a n/a

CONT.

721 BBN 9299 NO NO 90 M 2 5.5 2.3

CONT.

733 BBN 931 1 NO NO 93 M 3 37.75 2.3

CONT.

751 BBN 9329 NO NO 80 M 0 45.75 3.3

CONT.

762 BBN 9340 NO NO 94 F 2 21 2.3

CONT.

781 BBN 4205 NO NO 87 M 2 24 3.3

CONT.

786 BBN 9354 NO NO 85 M 2 30.5 3.3

CONT.

818 BBN_4229 NO NO 87 F 3 47 2.3

CONT.

826 BBN 9365 NO NO 86 F 2 32 3.4

Table 2

The AD cases all had a history of progressive dementia and were selected on the basis of a diagnosis according to CERAD of 'definite AD' and a Braak tangle stage of V-VI; according to NIA-Alzheimer's Association guidelines AD neuropathological change was considered a sufficient explanation for the dementia in all cases. The normal controls had no history of dementia, few or no neuritic plaques, and no other neuropathological abnormalities.

Immunohistochemistry

Double immunofluorescence and/or immunohistochemistry were used to assess the distribution of mCRP (mouse anti-human mCRP-specific antibodies 8C10 and p-IRS-1- Y1179) and activated microvessels (CD105/endoglin rabbit polyclonal antibody) as well as the presence of β-amyloid and p-tau (rabbit polyclonal antibodies). After incubation with primary antibodies for 1 h at room temperature (1 : 100), sections were washed and then incubated with the appropriate secondary antibodies (1 :50) - peroxidase (HRP), fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Jackson) or tetramethylrhodamineisothiocyanate-conjugated rabbit anti-goat (Jackson). Images were captured with Nikon 80i Digital Microscope using Nis Elements 3.21 software with multichannel capture option. Negative control slides were included where the primary antibody was replaced with PBS. Vecor ABC kits were used for all IHC and the Vector mouse on mouse (M.O.M) was used when applying mouse primary CRP antibodies to the murine brain sections with mouse secondary.

Statistical analysis

All in vitro experiments were performed at least twice and where appropriate, SPSS package was used with a student t test and ANOVA to determine statistical differences from minimum of n=3 groupings of test versus control. For the animal experiments, based on the advice of a medical statistician and the experience of behavioural studies of our team, n=8 was used for each group of mice providing the minimum number to allow significant data to be recognized.

Results

Kinexus quantitative phospho-protein screens demonstrated that mCRP increased phosphorylation of Tau and IRS-1 in BAEC

We performed a Western phospho-protein screen on BAEC exposed to mCRP C^g/ml, 8 minutes; based on our previous published findings of maximal acute phosphorylation induced by mCRP). Results demonstrated that Tau was phosphorylated (S516) by mCRP (>2 fold) and also IRS-1 (Y1 179) (>3 fold) amongst other proteins including focal adhesion kinase and Bcl2 (Figure 1A). Western blotting confirmed the results of the kinexus screen showing that IRS-1 and tau were phosphorylated in the presence of mCRP in BAEC after 8 minutes. Approximately a 4.5 fold increase in p-IRS-1 was found in BAEC exposed to mCRP for 8 minutes (Figure 1 B), and p-tau increased by approximately 4.2 fold (Figure 1C). The bar chart demonstrates the increase compared with control, untreated cells using β-tubulin as a house-keeping control. Since increased Tau phosphorylation, tangle formation and abnormal amyloid processing may be linked to vascular dysfunction in endothelium, it was examined if mCRP could affect/induce NFT formation, β-amyloid 1-42 cleavage or γ-secretase-presenilin expression in BAEC. The cleaved amyloid fragment (1- 42) was increased in samples (intracellular) treated with mCRP ^g/ml/24h) as shown by Western blotting (2.8 fold) (Figure 1 D). Extracellular levels of amyloid-β (1-42) were not significantly altered as measured in the medium (data not shown), γ-secretase active sub- unit (presenilin enhancer protein 2; PEN-2) and phosphorylated amyloid precursor protein (p- APP) expression was also increased around 2.5 fold after 8 minutes treatment (Figure 1 D) indicating a potential mechanism for amyloid cleavage. mCRP also phosphorylated ERK and AKT.

Down-regulation of IRS-1 with siRNA significantly inhibited the ability of mCRP to induce angiogenesis in BAEC

To confirm that IRS-1 was important in mediating the angiogenic properties of mCRP, down-regulated IRS-1 (>85%; Figure 2A) was down regulated using siRNA and this reduced the ability of mCRP to induce tube formation in a matrigel substrate by approximately 50% (Figure 2B; *=p<0.05 and **=p<0.01). Thus mCRP appears to operate through a pathway involving IRS-1 in order to mediate its pro-angiogenic activities. Similarly, addition of the mCRP antibodies (^g/ml) prior to addition of mCRP inhibited signalling through MAP kinase (ERK1/2 and IRS-1) and also perturbed angiogenesis following analysis of mCRP-induced tube-like structure formation, whilst the control IgG antibody showed no effect (Figure 2 C and D respectively).

mCRP induced increased vascular permeability with instability of cell-cell junctions and haemorrhagic angiogenesis in vivo

Electron microscope images of tube-like-structures produced in matrigel from a spheroid assay, in the presence of mCRP (10μg/ml; 24h) showed increased gaps between adjacent cells suggesting a possible increase in vascular permeability (Figure 3A). In addition, sprouts formed in the presence of mCRP were longer and thinner than those cultured in complete medium. Changes to the structure and/or organisation of the blood vessels was confirmed in vivo, with mCRP (10μg/ml applied to dorsal matrigel implants, inducing haemorrhage after 72h, unlike the normal pro-vascularization seen in the presence of VEGF (25ng/ml). Pentameric CRP produced no notable angiogenic response (n=3 and statistical comparison was not carried out with these implants) (Figure 3B). In addition, using a Millipore cell permeability assay, we showed that incubation of a BAEC monolayer cultured on collagen with 1C^g/ml mCRP (8h) induced a significant increase in cell permeability (p<0.01), and similar to that produced by pre-incubation with 10% DMSO (Figure 3C).

mCRP increased the expression of N-cadherin-often associated with inflammatory/unstable or aggressive angiogenesis (Figure 3D); but no other cell surface markers of EC activation/cell junction remodelling (ICAM-1 , VCAM, α₅β₃ integrin-data not included).

In vivo mouse study

Treatment with mCRP significantly deteriorated non-cognitive and cognitive behavior of none-transgenic mice. Intrahippocampal injection of CRP distinctly deteriorated several aspects of general behavior and cognitive responses tested in NTg mice. As positive controls, five-month old 3xTg-AD mice showed changes to their behavior and their capacity of learning and memory, as expected. mCRP did not decrease mouse reflex responses or coarse motor coordination on a rod, neither grip strength when clung on a wire. However, CRP reduced fine coordination significantly in NTg mice and as a trend in 3xTg mice, as shown in the wire hang test (Figure 4A; p<0.05). No significant changes were induced by CRP treatment on the freezing time, horizontal and vertical movements and emotionality behavior as tested in the open field test. CRP did not induce neophobia, anxiety or depression-like behavior in the NTg mice as tested in the corner test, dark and light box test and tail suspension test, respectively, nor increased the level of these behaviors in Tg mice. However, the treatment with CRP induced a significant effect in the animal behavior in the Boissier's hole board test, significantly decreasing the exploratory activity of NTg mice and further decreasing that of Tg mice (Figure 4B-C; p<0.05). mCRP induced cognitive loss in NTg mice, but cognition-related effects could not be detected in Tg mice because of their own low capacity of learning and memory. Lack of visual discrimination of a previously explored object induced by mCRP treated mice was detected in the novel object recognition test when object A was replaced by object B and C (Figure. 5A-C; p<0.05). Significant reduced acquisition of spatial learning and lack of retention of memory induced by mCRP were demonstrated in the MWM (Figure. 5D-F; pO.05). Histology

Following injection at CA1 hippocampal region, mCRP staining was identified along the retrosplenial ventral tract, lining of the dorsal 3^rd ventrical and within some surrounding major blood vessels and local cortical neurons (Figure 6Ai) (n=5) animals examined by histology in each group. All animals demonstrated the following similar features. Small microvessels were mCRP positive at the vicinity of injection site together with patchy areas of surrounding cortical neurons, especially in temporal association areas and primary auditory area, layer 5 and 6a. Scattered groups of neurons were mCRP-positive within cortex regions adjacent to injection site (Figures 6Ai-iii arrows), and in more distant cortical areas (primary and secondary motor areas, layers 2-5; Figure 6Aiv). There was a homogenous peri-nuclear staining in groups of neurons from the ipsilateral hippocampal region and cells appeared irregular and hypertrophic. The later staining was more evident within hippocampal field CA1 pyramidal layer, dentate gyrus molecular and granular layers, but not in field CA3 (Figure Av) Interestingly, there was also notable mCRP staining within hypothalamic neurons, posterior hypothalamic nucleus, lateral hypothalamic area and periventricular hypothalamic nucleus (Figure 6Avi).

Mouse cerebrum microvessels close to hippocampal formation were strongly positive for mCRP. Microvessels from local cortex regions were also positively stained (Figures 6Avii- viii) Distally, staining within microvessels was evident within the hypophysis region. Non- injected control animals showed no positive staining for mCRP in hippocampal or cortical regions (ix and x respectively).

In order to assess AD pathology, we performed p-Tau and Αβ immunostaining on serial sections of mice brains. CA1 pyramidal neurons and dentate gyrus molecular and granular layers were positively stained for p-Tau and Αβ (Figures 6BN and iii and 6Ciii respectively). There was significant p-Tau staining within the hippocampal CA3 region, shown positive for mCRP (Figures 6Biv-vi), whilst Αβ was primarily increased in sporadic cortical neurons and diffuse plaque-like elements associated with dying neurons (Figure 6Cii and iii), mimicking the pattern seen in 3 x Tg mice (Figure 6Civ and v)). P-Tau staining was generally more abundant within cortical areas, mainly in big pyramidal neurons both in ipsilateral and contralateral hemisphere, with notable axonal positivity. This was especially evident with ecthorinal area 5, layer 5 and temporal association areas, layer 6. Peri-nuclear and axonal staining was seen in piriform cortical neurons of coronal (Bregma -1.94mm) sections in the hemisphere of injection. The later neurons were oedematous with ballooned, vacuolized morphology. Within ipsilateral basal ganglia, there were positive p-Tau neurons in thalamus, mainly posterior complex and posterior lateral nucleus of thalamus.

Since we identified an increase in p-IRS-1 within vascular endothelial cells on exposure to mCRP, we examined its expression and localization within our mCRP-treated animals by IHC. Increased p-IRS-1 expression within cortical neurons was identified in the same areas of serial sections as mCRP-positive cells (Figure 6Di-ii), as well as ventricular tracts (Figure 6Diii-iv), hippocampal neurons (Figure 6Dv-vi), some cortical microvessels and cortical plaques (Figure 6Dvii-viii). No obvious staining was seen in cortical microvessels. Non- transgenic animals showed very little staining for p-IRS-1 (Figure 6Dix).

Double IF labelling demonstrated co-immunolocalization of hippocampal neurons and CA1 and dentate gyrus neurons with mCRP (TRITC) and p-Tau (FITC) (Figure 6Ei and ii respectively) as well as cortical microvessels mCRP (TRITC) and CD31 (FITC) (Figure 6Eiii).

Interestingly, serial sections from the same areas showed numerous mCRP positive medium sized microvessels that were also CD105-positive (Figure 6E iv and v). This was not seen in the contralateral hemisphere. Deep into the brain within periventricular areas of thalamus, including paraventricular nucleus of thalamus, lateral habenula, intermediodorsal nucleus and central medial nucleus of thalamus had medium sized microvessels that were CD 105 positive. The density of the above positive blood vessels was higher closer to hippocampal dentate gyrus with postive neurons for mCRP and p-Tau on serial sections. Analysis of negative control sections (normal wild-type mice injected with buffer only) showed very little staining of neurons for p-Tau or Αβ and no evidence of Αβ plaques as shown in the various images, whilst in contrast, positive control 3xT mice showed similar staining pattern to non-transgenic mCRP-injected animals but more widespread and more cortical diffuse staining with the appearance of amyloid plaques (See Figure 6A-E control images).

mCRP directly induced phosphorylation of potentially neurodegenerative signalling intermediates in cortical neurons

Western blotting demonstrated that rat cortical neurons cultured in basal medium for 24h prior to experimentation, exhibited a notable increase in expression of p-Tau and p- ERK1/2 following 8 minutes of exposure to mCRP (1 C^g/ml; Figure 7i). In addition, p-IRS-1 , p-AKT and p-APP were also increased in mCRP-treated cells (Figure 7ii). Pre-treatment with blocking mCRP-antibody as described above before addition of mCRP notably reduced the phosphorylation of both p-ERK1/2 and p-Tau (Figure 7iii). In order to gain a further understanding of possible signalling pathways activated by mCRP in neurons, a KINEXUS Western phospho-protein array was conducted and results from this indicated a possible increase in expression p-FAK, p38 and p53 whilst a reduction in expression of RSK-1 and ERK-5 was seen (Figure 7iv).

The aggregation of Tau244-372 was induced directly by addition of mCRP in vitro

When Tau was incubated with mCRP (10 μg/ml) over a period of 24h (time of incubation optimised from tests at 1 h-5 days in pilot studies not included), scanning electron microscopical analysis of sputter-coated air-dried samples revealed increased helical tau filament polymerisation (dimerization) and formation to around 100nm, whilst incubation with Arachidonic acid (150μΜ), used as a positive control produced a similar effect (Figure 8). Anatomopathological study

Localization and expression of mCRP in Alzheimer's brain tissue with and without ischaemic stroke: Pentameric CRP was almost un-detectable in any of the brain tissue samples. There was strong expression of mCRP in the stroke brains from patients with clinically confirmed dementia and neuropathology of AD (β-amyloid-positive plaques and, tauopathy). mCRP was observed in peri-infarcted and infarcted regions in plaques from all five stroke patients with AD and IS (Figure 9A; Table 1). mCRP positive neurons were also present in regions adjacent to the infarct but further away where the tissue appeared normal (i.e. no stroke tissue damage but positive for plaques and other features of neurodegeneration), the mCRP staining virtually disappeared. There was a weaker staining pattern within the infarcted core and particularly in old infarcts (14 days and beyond). Penumbral regions and other areas with strong tau/phospho-tau pathology showed the most intense staining for mCRP. The mCRP becomes strongly expressed in ischaemic microvessels (see Figure 9B;), was also associated with neurons from within the plaques (see Figure 9BN) as well as with plaque elements with the appearance of neurofibrillary tangles (see Figure 9C). Interestingly, in AD patients (without known stroke), we observed frequent regions of tissue damage with the appearance of small cortical lacunes, and these regions stained most strongly for mCRP. mCRP-positive areas clearly surrounded neuronal plaques, but the staining was negative in end-stage dead neurons.

In sections from patients with clear AD pathology but no evidence of tissue damage caused from lacunar stroke or other hypoxic conditions, there was some staining of mCRP in neuronal plaques. However, there was no microvessel mCRP localization, suggesting further, that, mCRP deposition in neurons and microvessels are independent. The presence of mCRP in plaques maybe related to mCRP involvement with AD pathophysoiology and de novo synthesis and not only due to deposition from leaky microvessels in tissue infarcted areas.

Observation of microvessels from within the infarcted and peri-infarcted regions also showed an intense staining of mCRP (FITC green) in β-amyloid-positive (Rhodamine-red) capillaries (see Figure 9D; Table 1). Arrows show areas of co-localization in confocal images. The increased microvessel mCRP staining was present also in vessels without amyloid, but staining was more prominent in sections with severe amyloid angiopathy. Vessels expressing mCRP were almost always CD105-positive suggesting activation and perhaps the potential to undergo angiogenesis (IHC staining with anti-CD105-DAB brown and IF TRITC red using anti-mCRP antibodies; see Figure 9E). No expression of mCRP was seen in the contralateral hemispheres of patients following ischaemic stroke (data not included). Figure 10i shows similarity between mCRP staining of cortical neurons and in a serial section, tau phosphorylation of the same region in stroke-affected cerebral cortex. In Figure 10ii, lacunar stroke micro-infarct core has been identified (arrow) and a gradual reduction in intensity of mCRP staining is seen as we move away from the damaged tissue region. This was common to all similar regions we examined.

Figure 11 shows a novel signalling pathway through which mCRP may contribute to pathological development of dementia. Key novel elements include IRS-1 , and NCAM.

Discussion

Epidemiological studies have demonstrated that approximately 25% of elderly patients show signs of dementia within 3 months of ischaemic stroke and that there is a significant increased risk of dementia associated with ischaemic stroke with specific relationships to cerebral hypoxia/ischaemia. Ischaemic stroke exacerbates dementia in Alzheimer^'s patients and animal models have demonstrated a strong relationship between neuroinflammation, increased platelet activation (which could involve mCRP -; and AD/stroke toxicity). In these cases, hypoxia is often associated with small vessel disease and vessel constriction or in- patency (vascular dementia; CAA). Vascular remodelling is a key feature of the neurodegenerative process, abnormal angiogenesis being strongly associated with β- amyloid deposition and the presence of NFTs in a study of post-mortem brain samples from AD patients, suggesting a relationship with tissue injury. Similarly, it has been shown using APP23tg mice and vascular casting, that vasculature often ended (was blocked) at the sites of developed amyloid plaques and surrounding hypoxic regions had tried to compensate by eliciting angiogenesis.

Since circulating pentameric CRP levels increase dramatically during cardio/cerebrovascular events and mCRP is now known to become tissue and cell associated as a monomer with strong biological properties, it made sense to investigate if it could be involved in modulating cell signalling associated with plaque development or vascular damage associated with small vessel disease in AD affected individuals. In this regard, only circulating pCRP concentrations (as pCRP is not found associated with tissue/cells) can be measured, which as stated previously range during active infection and prior to/during cardio/cerebro-vascular ischaemia between 40-20C^g/ml in patients. And hence the chosen mCRP delivery of between 5-1C^g/ml (in vitro) and-5C^g/total drip- (^g/minute) delivery comfortably fits within these limits and was designed to reflect this following our previous work and that of others showing no cytotoxicity to cells within this range of use.

In addition, our previous histological studies of ischaemic stroke patients allowed identification of the expression of mCRP in co-existing amyloid plaques and elements with the appearance of NFTs; however, where there was no evidence of previous ischaemic stroke or in regions of lacunar/silent strokes, very little mCRP was seen. In patients with AD but without evidence of stroke, mCRP was only evident by its relatively weak incorporation into some AD plaques neighbouring damaged neurons, suggesting an additional possible de novo mechanism of synthesis distinct from deposition through vascular leakage. Penatmeric CRP was not found to be expressed in any of the tissue samples.

The current experiments demonstrate a potentially key role for mCRP in patients with previous vascular or ischaemic brain damage (which could include brain trauma injury) in perpetuating dementia onset. Our four key elements for proof of principle are 1) mCRP induces abnormal angiogenesis, producing vessels in vitro allowing, greater permeability, and signalling activation reflecting a possible mechanism for perpetuating inflammation; 2) CA1 hippocampal injection of mCRP in a murine model of AD, directly induced cognitive and behavioural decline concomitant with AD-like brain structural changes including increased expression of p-Tau and β-amyloid plaque production; 3) mCRP induced tau filament polymerization in vitro and in addition, mCRP induced neuronal signalling pathways associated with AD pathological development; 4) a detailed histological study in patients with AD with and without stroke demonstrated a strong expression and co-localization of mCRP and associated signalling molecules with AD elements, with strong co-localization to areas of tissue disruption caused by infarct.

In evidence for the impact of mCRP on vascular function, we showed using tri- dimensional spheroids in combination with TEM that inter-cellular spaces or gap junctions between adjoining cells were notably greater in sprouts developing from mCRP-treated tridimensional spheroids. In addition, mCRP significantly increased the permeability of a confluent, barrier endothelial cell monolayer to FITC dextran and also produced haemorrhagic tissue lesions concomitant with angiogenesis following dorsal matrigel implantation in vivo (examined macroscopically only). This data suggests strongly that mCRP may induce an increased permeability of abnormally developing microvessels after tissue injury, and this could be linked to exacerbated inflammation and/or haemorrhage in the region if the same pattern were reproduced in vivo in developing or damaged microvessels. This could have relevance to vascular dementia and that linked to ischaemic stroke, where, the micro-environment existing in the vicinity of susceptible vessels may be unbalanced in the presence of mCRP leading to more aggressive but less efficient angiogenesis producing the same immature and weak vessel walls seen in tumour/plaque vascularisation.

We then investigated the potential signalling mechanisms through which mCRP might affect vascular formation and development using a specifically designed Western phospho- protein screening array with a particular focus on vascular activation pathways associated with neurodegenerative disease. Quantitative analysis of protein phosphorylation changes induced in BAEC in the presence of mCRP demonstrated that p-IRS-1 and p-tau amongst others were notably increased. Insulin growth factor and its substrates IRS-1/2 have been implicated in the vascular complications of diabetes associated with AD in association with β- amyloid clearance. The experiments show that siRNA knock-down of IRS-1 partially inhibited the pro-angiogenic effects of mCRP by blocking tube-like structure formation in matrigel and abnormal, fragile sprout formation in EC-generated spheroids. IRS-1 is now known to be strongly pro-angiogenic, and down-regulation of its expression blocks angiogenesis both in vitro and in vivo. Hence, this is one potential novel mechanism that mCRP may try to promote new vascular growth in angiogenic areas of damaged or stroke-affected areas of AD brain tissue. It is interesting to consider that the insulin-like growth factor-1 receptor (IGF-1 R) might be a candidate receptor for mCRP binding since it directly phosphorylates IRS-1 and its inhibition has also been shown to be sufficient to block angiogenesis.

mCRP was shown to induce and increase in expression of NCAM-a marker of immature endothelial cells and linked to active increased EC permeability, but did not affect VCAM, ICAM or integrin expression. Therefore, Tau phosphorylation/NCAM activation could be a mechanism through which mCRP increased the EC permeability.

The de novo modification/production of toxic amyloid could also be related to mCRP- induced endothelial dysfunction in vitro, in vivo, and potentially in vascular-based dementia. We investigated if the antibody to mCRP could potentially block the down-stream signalling activation and found that pre-incubation with anti-mCRP did significantly reduce angiogenesis and also signalling through p-ERK1/2 and p-IRS-1 with an ND₅₀ value of around ^g/ml (not kinetically determined).

The experiments showed that direct hippocampal injection of mCRP in mice resulted in both CA1-3 positively stained neurons as well as local microvascular staining. Neuronal cells stained concomitantly with p-Tau and cortical regions of microvessels positive for mCRP were also CD105 and p-IRS-1-positive (serial section analysis) suggesting a promotion of angiogenesis or vascular activation. mCRP became 'stuck' indefinitely in the ECM and was found in cortical, hippocampal and hypothalamic neurons, producing vacuolated and/or swollen cells (present and remaining more than 1-month after injection). No overall cell loss was observed, and control mice injected with equivalent concentrations of CSF protein showed no reaction nor signalling activation. Tau aggregates were seen localised to abnormal looking neurons and axons also became p-Tau-positive. In addition, β-amyloid-like plaques were present and positively stained for mCRP, and overall the pattern of pathological staining was not dissimilar to that produced in transgenic animals visualised by IHC/histology. Most importantly, animals showed behavioural and cognitive deficits similar to triple transgenic animals with induced progressive AD-like brain pathology, including novel object recognition failure, Morris water maze distance and time, and wire-hang testing. This provides the first evidence that mCRP laid down within the brain parenchyma (such as following brain infarction) might directly promote development of neurodegeneration. No further cognitive or behavioural decline was observed in 3 x Tg animals that were also injected with mCRP. In addition, mCRP was not detected in normal nor 3 x Tg mice even as a consequence of or concomitant with neurodegenerative pathway activation, suggesting it is not produced and/or deposited in this model as part of a normal inflammatory cascade, nor produced by de novo synthesis within the brain.

Since mCRP was found within neurons, the effects of this molecule on cortical neuronal signalling were examined using Western blotting and Kinexus phospho- microarrays. Similar to ECs, mCRP induced an increase in p-ERK1/2 and p-Tau expression, and in addition, p-APP, p-Akt and p-IRS-1 were stimulated within 8 minutes exposure. Once again, blocking mCRP-antibody incubation was sufficient to inhibit p-ERK1/2 and p-Tau signalling. Additional proteins increased on the microarray included focal adhesion kinase (FAK) and p53, both of which could influence signalling linked to Αβ-induced cellular apoptosis.

One of the hallmarks of AD is the fibrillization of tau protein wherein, individual monomers aggregate, particularly when hyper-phosphorylated eventually to form paired helical filaments and this can be visualised using S.E.M./T.E.M.. The experiments showed that tau oligomers produced evidence of tau filament assembly/aggregation in the presence of mCRP, and similar to that induced by arachidonic acid used as a positive control. Similar looking mCRP-positive staining fibrils were also identified within the murine-mCRP-injected brains and AD patients with stroke. Several studies have identified a correlation between neurotoxicity and cognitive dysfunction, tau aggregate expression preceding NFT formation in animal models, which leads to a potential hypothesis that mCRP could contribute to this pathological progression. For the experiments, the murine model as under inflammatory conditions was specifically chosen, CRP production is a minor part of the acute response to insult-and was not evident within the brains of our transgenic AD mice. Although stereotactic injection of pentameric CRP was not performed in the model used, it is likely that it would have undergone a similar fate within the brain on contact with cells and tissue dissociating to mCRP and producing a similar effect to the mCRP - this would be our hypothesis of how the majority of mCRP may build up within the damaged brain tissue in AD. A limitation of the use of the murine model here, is that it cannot be used effectively to identify the impact of mCRP for example after stroke or vascular injury/traumatic brain injury on pathophysiological AD progression, due to its lack of production as an acute phase response protein.

In AD patients, it was shown that expression of mCRP, but not pentameric CRP was expressed strongly in microvessels but only following ischaemic stroke and in stroke-affected regions. Vessels were also β-amyloid-positive in many cases suggesting the presence of small vessel disease and usually CD105-positive suggesting abnormal activation and perhaps angiogenesis. Cerebrovascular pathology is thought to be a key element associated with AD pathology and in particular, inhibition of angiogenesis, which may be an attempt to re-perfuse hypoxic areas of brain tissue, which may be attributed to β-amyloid deposition. Strong mCRP positivity was observed within plaques-again particularly in patients with ischaemic stroke, and in addition, neurons and NFTs in p-tau/Αβ strongly positive regions were also stained positive for mCRP. It is interesting that when we examined micro-infarcted areas or lacunar strokes from patients with AD, there appeared to be a gradient of expression of mCRP being strongest near to the infarct core decreasing to a weak expression approaching regions with the appearance of normal tissue structure. This was concomitant with a greater concentration of p-Tau staining and Αβ-positive material.

Therefore, mCRP may provide a causative link between ischaemic stroke, microinfarction/lacunar insult or traumatic brain injury associated with vascular damage and inflammation, and the significantly increased risk of development of dementia experienced within this population-based on the findings described. The present inventors believes that the production of small molecule inhibitors/antagonists or blocking antibodies could form the basis of a novel therapeutic strategy to inhibit down-stream processing linked to neurodegeneration/dementia after stroke.

Examples of mCPR modulating molecules

The following examples illustrate the range of potential therapies which could be developed for use in the treatment, prevention or management of neurological degeneration or dementia by inhibiting/modulating mCRP.

Antibody

A monoclonal antibody which has been raised against the VCLHFYTELSSTR (SEQ ID No. 2) epitope in a pharmaceutically acceptable carrier.

The production of monoclonal antibodies which reacts with an epitope that is associated with the lipid raft, cholesterol-binding domain of the mCRP molecule, which includes residues that involve the single intrachain disulphide bond of mCRP (i.e. residues C36 and C97 of the 206 aa mCRP molecule). The cholesterol binding domain of mCRP is more prominently reactive after the intrachain disulphide bond is reduced hence focus on the rmCRP- offering an allosteric activation mechanism that controls the bioactivity of rmCRP with cell membranes, and triggering inflammation.

As such these monoclonal antibodies would be focused on this molecular region (ss) of reduced rmCRP-cholesterol binding region (residues 35-47 of the cholesterol-binding site), and could be could be used as a blocking antibody that can control the function of mCRP as a potent pro-inflammatory signal.

Peptide

A peptide comprising an amino acid sequence which is able to bind to VCLHFYTELSSTR (SEQ ID No. 2) in a pharmaceutically acceptable carrier.

Small molecule

A small organic molecule which mimics the binding properties of the above peptide in a pharmaceutically acceptable carrier. Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1. A composition for use in the treatment, prevention or management of neurological degeneration or dementia, comprising an inhibitor or antagonist of monomeric C- reactive protein (mCRP), and optionally, wherein the inhibitor or antagonist modulates the activity of the cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP.

2. The composition of claim 1 , wherein the inhibitor or antagonist binds to amino acid residues of mCRP at amino acid residues of about 35 to about 47 and/or about 199 to about 206.

3. The composition of either claim 1 or 2, wherein the inhibitor or antagonist binds to at least part of the amino acid sequence VCLHFYTELSSTR (SEQ ID No. 2) and/or FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% homology thereof.

4. The composition of any preceding claim, wherein the inhibitor or antagonist comprising a peptidic moiety according to a Formula 1 or salt, derivative, prodrug or mimetic thereof:

Formula 1 : [X1-X2-X3-X4-X5-X6]

wherein X1 may be present or absent, when X1 is present, X1 comprises V; wherein X2 may be present or absent, when X2 is present, X2 comprises C; wherein X3 comprises L;

wherein X4 comprises H, R, G, W or Y;

wherein X5 comprises up to 170 amino acids; and wherein X6 may be present or absent, when X6 is present, X6 comprises FTKPQLWP.

5. The composition of claim 4, wherein X5 comprises C at position 59 if X2 is present.

6. The composition of claims 4 or 5, wherein X1 to X5 comprises VCLHFYTELSSTR.

7. The composition of claims 4 to 6, wherein the composition comprises amino acid residues or amino acid analogues.

8. The composition of claims 4 to 6, wherein the composition comprises a peptide or peptide mimetic molecule.

9. The composition of any preceding claim, wherein the inhibitor or antagonist comprises an antibody or antibody mixture.

10. The composition of any preceding claim, wherein the neurological degeneration or dementia is related to or due to inflammation.

1 1. The composition of claim 10, wherein the inflammation is the result of neurovascular or traumatic event or to acute or chronic hypoperfusion.

12. The composition of any one of claims 1 to 9, wherein the neurological degeneration or dementia is related to or due to thrombotic or embolic vessel occlusion.

13. The composition of any one of claims 1 to 9, wherein the neurological degeneration or dementia is related to or due to an ischaemic event.

14. An antibody or antibody mixture, for use in the treatment, prevention or management of neurological degeneration or dementia, wherein the antibody or antibody mixture is capable of binding to the cholesterol binding region (CBR), and optionally, to the C- terminal octapeptide region of monomeric C-reactive protein (mCRP).

15. The antibody or antibody mixture of claim 14, wherein the antibody binds to mCRP between or at amino acid positions of about 35 to about 47 and optionally within about 199 to about 206.

16. The antibody or antibody mixture of either claim 14 or 15, wherein the antibody binds to at least part of the amino acid sequence VCLH FYTELSSTR (SEQ ID No. 2) and optionally FTKPQLWP (SEQ ID No. 3) of mCRP or derivative sequences having at least 75% homology thereof.

17. A composition for use in the treatment, prevention or management of a neurodegenerative condition comprising an antibody or peptide analogue thereof which binds to monomeric C-reactive protein (mCRP), wherein optionally the antibody or peptide analogue thereof modulates the activity of the cholesterol binding region (CBR) and/or the C-terminal octapeptide region of mCRP, and wherein the composition is administered in a therapeutically effective amount:

(a) after an ischaemic event;

(b) after a thrombotic or embolic vessel occlusion;

18. The composition of claim 17, wherein the composition is administered up to about 72 or 48 hours after an ischaemic event.

19. The composition of claim 17, wherein the composition is administered during or immediately after an ischaemic event.

20. The composition of claim 17, wherein the composition is administered prophylactically to individuals having elevated levels of mCRP.

21. The composition of any one of claims 17 to 20, wherein the composition is administered intra-arterially.

22. A kit of parts comprising the composition according to any one of claims 1 to 13 or the antibody or antibody mixture according to claims 14 to 16, a receptacle in which said composition or antibody or antibody mixture is provided and equipment required for the administration of said composition or antibody or antibody mixture.

23. The kit of claim 22, wherein the equipment comprises intra-arterial delivery device.