FIELD OF THE INVENTION

The invention relates to treatments for cognitive decline and/or memory impairment based on reducing the extracellular matrix in the hippocampus. Improved compositions are provided for such treatments. The invention also relates to detection methods for the early diagnosis of cognitive decline and/or memory impairment based on an increase in extracellular matrix in the hippocampus. The treatments and diagnostic methods are especially useful in the field of Alzheimer's disease.

BACKGROUND OF THE INVENTION

With the quality of life in developed countries continuing to improve, so does the proportion of aged individuals in our population. The prevalence of age- related health problems is expected to rise concomitantly with our increasing lifespan. In particular, aged individuals have to cope with an age-related decline in memory function and cognitive performance, which may seriously affect their quality of life. A better understanding of the neurobiological mechanisms underlying age-related cognitive decline is crucial, to facilitate maintenance of cognitive health in the elderly, and to reveal potential causes of highly prevalent age-related sporadic forms of Alzheimer's disease in which cognitive decline is severely impaired by yet unknown mechanisms. There is currently a need for improved treatments and methods for the early diagnosis of cognitive related disorders. Alzheimer's disease (AD) is a progressive age-dependent neurodegenerative disorder and the most common cause of dementia in the elderly. AD is characterized by global and progressive cognitive decline, causing impairments in memory processing, judgment and thinking, all of which eventually interfere with daily living. Neuropathological hallmarks of AD are the presence of diffuse and neuritic plaques, which are mainly composed of 6- amyloid (A6) 39-42-amino-acid peptides formed after proteolytic cleavage of the β-amyloid precursor protein (APP) by 6- and γ-secretases, and of

neurofibrillary tangles which are intraneuronal aggregates of

hyperphosphorylated tau protein (Braak, de Vos et al. 1998; Selkoe 2001). The currently prevailing hypothesis on the etiology of the disease is the amyloid cascade hypothesis, which states that overproduction of A6 is a necessary prerequisite for the neuropathological and clinical changes observed in AD (Hardy and Higgins 1992; Karran, Mercken et al. 2011). Yet, early memory impairments in human AD patients are not temporally correlated with the formation of Αβ plaques in brain areas that are important for memory processing, such as the hippocampus. Further insight into what causes cognitive decline in AD is thus necessary in order to develop effective treatments and methods for early detection.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a method of treating an individual having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia, comprising administering to an individual in need thereof one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual.

In some embodiments, one or more compounds are provided that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of an individual for use in the treatment or prevention of age- dependent cognitive decline and/or memory impairment, or a dementia.

Preferably, said one or more compounds treat at least one cognitive or memory related symptom of said dementia. Preferably, the alteration of the amount of or the composition of the ECM in the hippocampus increases synaptic plasticity in the hippocampus.

Preferably, at least one of the one or more compounds comprises a nucleic acid encoding a membrane-anchored protein that alters the amount of or the composition of the extracellular matrix.

In a further aspect of the disclosure, an isolated nucleic acid encoding a recombinant membrane-anchored protein is provided, wherein said

recombinant protein alters the amount or the composition of the extracellular matrix (ECM) in the hippocampus.

In a further aspect of the disclosure, a method of identifying a compound for treating an individual having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia, comprising administering to a rodent a one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus and determining the affect on cognitive decline and/or memory impairment.

Preferably, said rodent is a mouse model of Alzheimer's disease.

In preferred embodiments of the disclosure, said one or more compounds alter the amount of or the composition of an ECM protein or hyaluronic acid.

Preferably the ECM protein is selected from a chondroitin sulfate

proteoglycans, more preferably aggrecan, brevican, neurocan, NG2, versican and phosphacan, more preferably from versican.

Preferably, said one or more compounds in not a metalloprotease or a metalloprotease inhibitor. Preferably, said one or more compounds is not MMP-9, MMP-2, MT1-MMP or an inhibitor of TIMP-1. Preferably, said compound is not a chondroitinase. Preferably, said compound is not a CD44 inhibitor.

In preferred embodiments of the disclosure, the impairment in memory or cognition is due to excessive ECM. Preferably, the dementia is Alzheimer's disease (AD). Preferably, the AD is in the early stages of AD, i.e., significant Αβ plaques have not yet accumulated in the hippocampus of said individual. In preferred embodiments of the disclosure, said one or more compounds increase the degradation of the ECM (preferably an ECM protein or hyaluronic acid as described herein) in the hippocampus and/or decrease the production of the ECM (preferably an ECM protein or hyaluronic acid as described herein) in the hippocampus and/or decrease the amount of at least one ECM component (preferably an ECM protein or hyaluronic acid as described herein) the ECM of the hippocampus.

In a further aspect of the disclosure, a method of classifying an individual as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia is provided, comprising determining the level of one or more extracellular matrix (ECM) components in the

hippocampus of said individual, wherein an increased level of ECM

components as compared to a reference indicates that said individual has or is at risk of developing a cognitive, memory, or neurodegenerative disease, wherein said ECM component is not Tenasin C.

In a further aspect of the disclosure, a method of classifying an individual as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or dementia is provided, comprising determining the level of two or more extracellular matrix (ECM) components in the

hippocampus of said individual, wherein an increased level of ECM

components as compared to a reference indicates that said individual has or is at risk of developing a cognitive, memory, or dementia.

In preferred embodiments of the disclosure, the ECM component whose level is determined is an ECM protein, preferably selected from Versican and CD44. In preferred embodiments of the disclosure, the level of one or more ECM components in the hippocampus is determined by detecting one or more ECM components in the hippocampus, preferably using non-invasive detection.

Preferably said method comprises administering to said individual a positron emission tomography (PET) compatible tracer, wherein said tracer binds to said one or more ECM components; carrying out a PET scan on said individual; and determining whether the hippocampus of said individual has an increased level of said ECM component as compared to a reference value. In preferred embodiments of the disclosure, the level of one or more ECM components in the hippocampus is determined by detecting one or more ECM components in a biological sample from said individual, preferably from blood, serum, or cerebrospinal fluid. Preferably, said method comprises performing an immunoassay to determine the level of one or more ECM components in the hippocampus. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic representation of the 8-plex iTRAQ approach. In each 8-plex iTRAQ experiment, combined left and right hippocampi of mice from of eight different ages were pooled and labeled with one of the eight iTRAQ labels. After labeling the samples were pooled and subjected to MS/MS analysis for identification and quantification of proteins. In total, eight replicate 8-plex experiments were performed.

Figure 2. Proteomic analysis identifies 25 proteins that are

significantly regulated over time. A, K-means clustering revealed four different expression clusters. Clusters 1-3 contained proteins whose expression was increased or decreased already early during aging, and remained high or low over time. Only proteins in cluster 4 showed progressive upregulation over time. These proteins also showed the strongest correlation between expression and age, as evidenced by R correlation coefficients. Average protein expression was calculated against 20 weeks. B, Temporal expression patterns of proteins in cluster 4. Three out of the four proteins in this cluster represent protein constituents of the ECM. Error bars represent SEM. C, Western blotting confirmed the age-dependent upregulation of each of these ECM proteins. * p<0.05, error bars represent SEM. Figure 3. Proteomic analysis identifies 101 proteins that are

significantly regulated at any time point. A, SAM analysis revealed 101 proteins being significantly differentially expressed at any time point. B, These proteins were divided in age groups: early-aged, middle-aged, old-aged.

Significantly upregulated proteins were almost equally present in all three age groups, whereas a progressive increase with age was observed for significantly downregulated proteins. C, All differentially expressed proteins were categorized in 17 functional synaptic protein groups as previously defined (Lips, Cornelisse et al. ; Ruano, Abecasis et al. 2010). Protein groups indicated in bold print were significantly enriched in that particular set of regulated proteins (p<0.05; see also Table 1). D, Individual protein expression profiles of significantly enriched functional protein groups. Red indicates proteins that are upregulated; blue indicates proteins that are downregulated. Figure 4. Variance in synaptic protein expression increases with age.

A, Distributions of the standard deviations (SD) of the average standardized protein intensities (iTRAQ peak areas) per time point revealed a significant increase over time (original data). B, Distributions of average standardized protein intensities (iTRAQ peak areas) per time point revealed no dependence on time (original data). C, Distribution of all SD values from all time points indicating the highly-constrained proteins as the top 5% low variability proteins (yellow box), and lowly-constrained proteins as the top 5% high variability proteins (green box) (original data). D, Functional enrichment analysis for high variability and low variability proteins at old-age using GO terms and KEGG pathway databases.

Figure 5. Overview of protein expression and stochasticity changes with aging. Low variability proteins (yellow box), high variability proteins (green box), upregulated proteins (red box). Figure 6. Identification of age-related changes in protein composition in 3xTg-AD AD mouse model. A, Schemtaic representation of the 8-plex iTRAQ approach. B, Proteomics analysis identified a total of 376 proteins. SAM analysis indicated that the levels that the levels of 104 proteins were significantly different (FDR < 10, log-fold change >0.125) between AD mice and wildtype controls at one or more time points. C, Most proteins were significantly regulated at 3 and 12 months of age.

Figure 7. Increased expression of Αρρ/Αβ and ApoE in APP-PS1 mice. A, ApoE expression was only significantly upregulated at 12 months of age. B, This could be confirmed by immunoblotting. * FDR <10 (iTRAQ) or p <0.05. C, App expression was significantly increased at 3 and 6 months of age. D, the additional increase observed at 12 months was primarily due to an increase of the specific A6 peptide and could be confirmed by immunolbotting. * FDR <10 (iTRAQ) or p <0.05. E, No plaques were observed at 3 months of age, whereas at 6 months, all APP -PS 1 mice had few hippocampal plaques and plaque load steadily increased up to 12 months. Parallel to the increase in plaque formation, there was a moderate increase in GFAP staining around all plaques at 12 months of age.

Figure 8. Functional overrepresentation of significantly regulated proteins. A, Proteins were assigned to one of the 17 functional synaptic protein groups as previously defined (Lips, Cornelisse et al. ; Ruano, Abecasis et al. 2010). B, Overrepresentation of differentially expressed proteins within functional groups was determined by calculating the difference between proportions and the ratio of proportions. Overrepresentation was only considered relevant when overrepresented functional groups contained at least 3 proteins, and when the ratio of proportions was >1.1. C, Overrepresented groups at 3 months of age were proteins involved in cell

adhesion /transsynaptic signaling, enalocytosis, excitability, exocytosis, and G- protein relay. Overrepresented groups at 12 months of age were proteins involved in cell adhesion/ transsynaptic signaling, cell metabolism, exocytosis, and G-protein relay. Figure 9. Age-related changes in the extracellular matrix in APP-PSl mice. A, There was an early (3 months) higher level of extracellular matrix proteins in young AD mice compared to wildtype controls, while expression levels returned to wildtype levels at older ages (12 months). B, This could be confirmed by immunoblotting. * FDR < 10 (iTRAQ) or p < 0.05.

Figure 10. Increased hippocampal staining of ECM in APP-PSl mice at 3 months of age. A, Using immunohistochemistry we could determine increased immunofluorescence for WFA and Tnr in AD mice compared to their wildtype controls, indicating an increase of extracellular matrix proteins in these animals at 3 months of age. (DAPI: blue; Tnr: red; WFA: green). B,

Expression of WFA-positive neurons was particularly high in the subiculum and the adjacent tip of the CAl region (DAPI: blue; WFA: green).

Figure 11. Reduced spatial memory in 3 months old APP-PSl mice. Hippocampal-dependent memory in APP-PSl mice and their wildtype controls at 3 months of age using a contextual fear memory task. A, scheme. B, wildtype and APP-PSl mice exhibited similar baseline activity during the training trials before the shock delivery. C, memory performance, as assessed by freezing behavior on re-exposure of mice to the context in which they were previously shocked, was reduced in APP-PSl mice compared with wildtype mice. * p < 0.05. Figure 12. Improved memory after ECM treatment. Three months-old APP-PSl animals and WT littermates were tested in a contextual fear memory paradigm (Control) and compared to a group of three months-old APP-PSl animals and WT littermates who received received a single dose of ChABC (ChABC).

Figure 13. Proteomics data showing increased levels of proteins in the early stage of AD.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENTS

Aging of the brain invariably leads to functional decline and impairments in cognitive performance and motor skills. Several studies suggested that normal brain aging is associated with subtle morphological and functional alterations in specific neuronal circuits, and does not include overall neurodegeneration in the brain (Rapp and Gallagher 1996; Rasmussen, Schliemann et al. 1996). Diminished cognitive function with increasing age is likely due to synaptic dysfunction and ineffective neurotransmission (Burke and Barnes 2006;

Barnes 2011). For instance, increasing evidence is in support of age-related alterations in hippocampal activity that co-occur with deficits in learning and memory in healthy aging humans (Daselaar, Fleck et al. 2006; Dennis, Hayes et al. 2008; Beeri, Lee et al. 2009). In addition, rodent models of healthy aging demonstrate strong correlations between impaired performance of aged rats in behavioral tests of hippocampus-dependent learning and memory on one hand, and disturbed hippocampal neuron ensemble activity on the other (Barnes, Suster et al. 1997; Wilson, Ikonen et al. 2003; Wilson, Ikonen et al. 2004;

Gerrard, Burke et al. 2008). Electrophysiological studies provide additional evidence that age-related disturbances in the hippocampus involve changes in the principle cellular features of learning and memory, i.e., synaptic long-term potentiation (LTP) and long-term depression (LTD) (Landfield and Lynch 1977; Barnes 1979; Norris, Korol et al. 1996; Barnes, Rao et al. 2000).

Together, these observations suggest that age-related decline in hippocampal synaptic efficacy and plasticity likely plays a critical role in cognitive

impairment.

Although the exact mechanisms underlying brain aging remain to be fully determined, they likely include changes at the molecular, cellular, and neuronal network levels. In particular, alterations in the molecular

composition and dynamics of hippocampal synapses could potentially reveal important aspects about the underlying mechanisms of brain aging. Age- related changes in global hippocampal gene and protein expression have been investigated previously (Blalock, Chen et al. 2003; Poon, Shepherd et al. 2006; Weinreb, Drigues et al. 2007; Loerch, Lu et al. 2008; Walther and Mann 2011), but were not geared to identify the specific synaptic molecular substrates of brain aging. Global synaptic protein expression studies have identified some of the molecular changes in hippocampal synapses, but interpretation of these data is hampered by the limited numbers of proteins that were analyzed and/or the limited temporal resolution of the aging time series that was used (Sato, Yamanaka et al. 2005; VanGuilder, Yan et al. 2010).

Aging is also the primary risk factor for Alzheimer's disease (AD), which clinically manifests as severe and accelerated age-dependent cognitive decline (Forstl and Kurz 1999). The degree of cognitive decline in patients with AD has been correlated with changes in the presynaptic vesicle protein

synaptophysin in the hippocampus and the association cortices (Dickson, Crystal et al. 1995; Sze, Troncoso et al. 1997; Reddy, Mani et al. 2005). In addition, downregulation of synaptophysin could also indicate a loss of presynaptic boutons as seen during later stages of AD. Previous studies have shown that as many as 45% of presynaptic boutons were lost in AD patients when compared to cognitively normal controls, especially in the neocortex and hippocampus, which may play a role in cognitive impairment in AD (Terry, Masliah et al. 1991; Masliah, Mallory et al. 2001). Developmental and activity- dependent plasticity of the ECM are known to be important in the regulation of synaptic function. During postnatal maturation of neuronal circuits, for instance, a developmental increase in ECM content corresponds with the ending of the critical periods in which synaptogenesis and synaptic refinement (Blue and Parnavelas 1983), myelin a lion (Ishiguro, Sato et al. 1991; Bruckner, Grosche et al. 2000) and the maturation of the nervous system (Pizzorusso, Medini et al. 2002; Carulli, Rhodes et al. 2006; Galtrey, Kwok et al. 2008) occur. In the adult brain, ECM components are known regulate synaptic plasticity in several ways (Pizzorusso, Medini et al. 2002; Gogolla, Caroni et al. 2009).

ECM proteins are highly expressed in the brain and are produced by both neurons and glial cells. During postnatal maturation of neuronal circuits these ECM pack into netlike structures, termed perineuronal nets (PNN), localized around a subset of neurons. The main component of ECM in the brain is polysaccharide hyaluronic acid. It acts as a backbone to engage proteoglycans and other glycoproteins into ECM structures. These protein components of the hyaluronic-based ECM are chondroitin sulfate proteoglycans (CSPGs; e.g., aggrecan, brevican, neurocan, NG2, versican and phosphacan), tenascins (e.g., Tnc and Tnr), and so-called hyaluronan link proteins (e.g., Haplnl) (Bandtlow and Zimmermann 2000; Yamaguchi 2000; Rauch 2004). Agrin is a heparan sulfate proteoglycan component of the extracellular matrix. CD44 is a cell surface receptor with its principle ligand being hyaluronic acid (HA) (Ponta, et al., Nature Rev. Mol. Cell. Biol. 2003, 4, 33-45; herein incorporated by reference). Additional components of the ECM include perlecan, laminin, fibronectin, collagen, pentraxins, pleiotrophin/HB-GAM, reelin,

thrombospondin.

Regulation of the ECM includes not only the production of new ECM but also controlling the rate at which the ECM is degraded. Hyaluronidases are a family of enzymes which degrade hyaluronic acid. Matrix metalloproteases

(MMP) are enzymes involved in the proteolysis of the ECM, which themselves can be inhibited by the tissue inhibitor of metalloproteinase family (TIMPl, -2, -3, and -4). Neurotrypsin is a brain-specific serine protease responsible for agrin cleavage. Tissue-type plaminogen activator (tPA) is a serine protease. It has been demonstrated that application of tPA to the brain has effects on synaptic plasticity.

The inhibition of CSPG is also an important means to affect the ECM.

Chondroitinase ABC (ChABC) is an enzyme that cleaves glycosaminoglycan side chains from a protein core. CSPGs are involved in inhibiting synaptic plasticity while treatment with ChABC after spinal core injury and reduce such inhibition (reviewed in Busch and Silver, Current Opinion in

Neurobiology 2007, 17: 120-127). CSPG function can also be affected by disrupting the glycosylation of CSPG. Such glycosylation is carried out by

Xylosytransferase-1 (XT-1). Inhibition of XT-1, for example via a DNA enzyme, reduces GAG chains (Hurtado et al. Brain 2008 131: 2596-2605). Such a strategy promoted spinal cord repair. Antibodies that bind sulphation motifs of CSPGs have also been demonstrated to inhibit CSPG function (Gama et al. Nat. Chem. Biol. 2006 2:467-473, the disclosure and in particular the antibodies described therein are hereby incorporated by reference). The synthesis of CSPGs can also be affected by inhibiting enzymes such as chondroitin 4 sulphotransferase. Lysyl oxidase (LOX or protein-lysine 6-oxidase) is an enzyme that cross-links collagens or elastins in the ECM. β-aminopropionitrile (BAPN) is an

irreversible inhibitor of LOX that has been used to reduce breast cancer metastasis. The developmental pattern of ECM formation corresponds with the ending of the critical periods in which synaptogenesis and synaptic refinement (Blue and Parnavelas 1983), myelination (Ishiguro, Sato et al. 1991; Bruckner, Grosche et al. 2000) and the maturation of the nervous system (Pizzorusso, Medini et al. 2002; Carulli, Rhodes et al. 2006; Galtrey, Kwok et al. 2008) occur. The ECM, therefore, is thought to limit developmental plasticity in various cortical areas. In the developing visual cortex, for instance, the absence of ECM during the critical period is considered a critical factor that allows ocular dominance plasticity to occur (Hensch 2005). Consistent with this notion, degradation of the ECM in the adult visual cortex restores ocular dominance plasticity (Pizzorusso, Medini et al. 2002).

It was recently reported that increases in ECM in late stage AD served as a protective mechanism to preserve synapse integrity (Lendvai et al. Acta Neuropathol 2012 Sep 9. [Epub ahead of print]). In contrast, the results described herein suggest that ECM expression limits hippocampal synaptic plasticity in early AD. The disclosure demonstrates the upregulation of proteins involved in the formation of extracellular matrix in APP-PS 1 mice as well as in otherwise healthy aged mice. The disclosure also demonstrates that modulation of the ECM, in particular by decreasing the ECM, can improve memory function in a mouse model of Alzheimer's (see e.g., Figure 12). In addition, the disclosure demonstrates that components of the ECM are also upregulated in the hippocampus of patients with Alzheimer's disease, including those in early stages of the disease. Accordingly, in one aspect the disclosure provides a method of treating an individual having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia, comprising administering to an individual in need thereof or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual. Treatment of the dementia refers to reducing or alleviating at least one symptom related to cognitive decline and/or memory impairment associated with said dementia.

As used herein, age dependent cognitive decline and/or memory impairment refers to those processes that generally progress more rapidly with age in healthy individuals. The term is used to distinguish from cognitive decline or memory impairment associated with, for example, head trauma, drug or alcohol misuse, infection, certain medications, thyroid disorders, or vitamin deficiencies. Preferably, said individual suffers from mild cognitive impairment (MCI). Dementia may also occur in younger individuals and includes

Alzheimer's disease, vascular dementia, frontotemporal dementia, and semantic dementia.

Memory impairment refers to the impaired ability to learn new information or to recall previously learned information. Cognitive decline includes aphasia (language disturbance); apraxia (impaired ability to carry out motor activities despite intact motor function); agnosia (failure to recognize or identify objects despite intact sensory function); and/or a disturbance in executive functioning, i.e., planning, organizing, sequencing, abstracting. The assessment of memory and cognitive disorders is well known in the art. See, e.g., "Diagnostic and Statistical Manual of Mental Disorders", 4.sup.th Edition (DSM-IV), American Psychiatric Association, Washington, D.C. (1994). Memory tests known to one skilled in the art include the Mini Mental Status Examination (MMSE), the Mini Assessment of Cognition (Mini-COG), the General Practitioner Assessment of Cognition (GPCOG) and the Six Item Cognitive Impairment Test (6CIT).

In a preferred embodiment, the dementia is Alzheimer's Disease. Typically, Alzheimer's Disease (AD) is diagnosed using a combination of clinical and pathological assessments. For example, progression or severity of AD can be determined using: Mini Mental State Examination (MMSE) as described by Mohs et al. Int Psychogeriatr 8: 195-203 (1996). PET scans are also used in conjunction with mental status testing. Preferably, an individual is administered a compound as described herein during the early stages of cognitive decline, such as the mild stages of

Alzheimer's disease. Symptoms of mild Alzheimer's disease include memory loss for recent events, changes in personality, difficulties in expressing thoughts, and misplacing items or getting lost. Preferably, a composition as described herein is administered to an individual before amyloid plaques have developed in the hippocampus.

Preferably, the alteration of the amount of or the composition of the ECM in the hippocampus increases synaptic plasticity in the hippocampus. Preferably, the alteration of the amount of or the composition of the ECM in the

hippocampus slows or prevents the progression of cognitive decline and/or memory impairment. It is within the purview of a skilled person to recognize whether a treatment slows or prevents the progression of cognitive decline and/or memory impairment in a particular individual as compared to the normal progression of such disorder in an untreated population.

In preferred embodiments, said or more compounds decrease the ECM in the hippocampus by at least 10, 20, 30, 40, or 60%. It is clear to a skilled person that the ECM should not be completely ablated, but rather reduced. For example, a combination of both chondroitinase and hyaluronidase would be expected to completely ablate the ECM functionally. Such treatment would be expected to negatively affect memory. Preferably, said or more compounds do not affect the ECM outside of the hippocampus. Such localized effect may be the result of, for example, localized administration to the hippocampus and/or targeting the one or more

compounds to the hippocampus (for example expressing said compounds under the control of a hippocampal specific promoter). Preferably, said individual is a mammal, more preferably a human.

Compounds which alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual include those which decrease the amount or composition of ECM proteins and/or hyaluronic acid. Preferably, said compounds decrease the amount or composition of chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; TIMP1; TIMP2; TIMP3;

TIMP4; CD44, lysyl oxidase; perlecan; laminin; fibronectin; collagen; tenascins (such as Tenascin-C and Tenascin-R); pentraxins; pleiotrophin/HB-GAM;

hyaluronan link proteins (e.g., Haplnl); reelin; thrombospondin; heparin- sulfate proteoglycan agarin; Xylosytransferase-1; and chondroitin 4

sulphotransferase. More preferably said compounds decrease the amount or composition of chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; CD44, and hyaluronan link proteins (e.g., Haplnl). More preferably said compounds decrease the amount or composition of chondroitin sulphate proteoglycans. Such decrease in amount or composition can include a reduction in the expression or secretion of an ECM protein or hyaluronic acid or the increase in degradation. It also includes a disruption in the post-translational

modifications of an ECM protein. Preferably, said compounds decrease the amount or composition of one or more chondroitin sulphate proteoglycans. Preferably, said amount or composition is altered using a ChABC or an enzyme capable of cleaving glycosaminoglycan side chains from a protein core.

The disclosure further provides pharmaceutical compositions for altering the amount of ECM or the composition of the ECM in the hippocampus useful in methods described herein comprising the one or more compounds and a pharmaceutically acceptable excipient.

Preferably, the compound is an inhibitor of one or more of the following, chondroitin sulphate proteoglycans (preferably selected from aggrecan, brevican, neurocan, NG2, versican or phosphacan); hyaluronic acid; TIMP1; TIMP2; TIMP3; TIMP4; CD44 (e.g., an inhibitory anti-CD44 antibody), lysyl oxidase; perlecan; laminin; fibronectin; collagen; tenascins (such as Tenascin-C and Tenascin-R); pentraxins; pleiotrophin/HB-GAM; hyaluronan link proteins (e.g., Haplnl); reelin; thrombospondin; heparin-sulfate proteoglycan agarin;

Xylosytransferase-1; and chondroitin 4 sulphotransferase; and/or a polypeptide or a functional fragment thereof or a nucleic acid encoding said polypeptide or functional fragment thereof selected from chondroitin as e ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue-type plaminogen activator (tPA), an antibody that bind sulphation motifs of CSPGs.

Preferably, the compound decreases the function or expression of one or more chondroitin sulphate proteoglycans (CSPGs). Preferably the compound is a polypeptide or a functional fragment thereof or a nucleic acid encoding said polypeptide or functional fragment thereof selected from chondoitinase ABC, an antibody that bind sulphation motifs of CSPGs, an inhibitor of

Xylosytransferase-1 (XT-1), and an inhibitor of chondroitin 4

sulphotransferase. Preferably, said ChABC is a mammalian ChABC, more preferably a human ChABC. The ChABC can be any enzyme having

chondroitin-sulfate-ABC endolyase activity.

In a preferred embodiment, the compound promotes the degradation or destabilzation of the ECM. Such compounds are known in the art and in preferred embodiments said compound is a polypeptide or a functional fragment thereof or a nucleic acid encoding said polypeptide or functional fragment thereof selected from chondroitinase ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue-type plaminogen activator (tPA), an antibody that bind sulphation motifs of CSPGs; or an inhibitor of hyaluronic acid, or an inhibitor of lysyl oxidase, TIMP1, TIMP2, TIMP3, or TIMP4.

In a preferred embodiment, the compound decreases the production of the ECM, in particular decreases the production of ECM proteins and/or decreases post-translational modification thereof. Such compounds are known in the art and in preferred embodiments said compound is an inhibitor of perlecan, laminin, fibronectin, collagen, xylosytransferase-1, chondroitin 4

sulphotransferase, tenascins, such as Tenascin-C (Tnc) and Tenascin-R (Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl); thrombospondin, heparin-sulfate proteoglycan agarin, and a chondroitin sulphate proteoglycans (CSPGs), preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan.

Preferred hyaluronidases include HYAL1, HYAL2, HYAL3, and PH- 20/SPAM1. Preferred metalloproteinases include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMPl l, MMP12, MMP13, MMP14, MMP15, MMP16, MMP23, and MMP24, more preferably MMP9

Preferably the compound is ChABC or an inhibitor of V-can or Tenascin-C. Preferably the compound is ChABC or an inhibitor of V-can.

Preferably the compound is not Tenascin-C and/or a lysyl oxidase inhibitor. Preferably, the compound is not ChABC or a CD44 inhibitor. Preferably, the compound is not Tenascin-C, a lysyl oxidase inhibitor, ChABC, or a CD44 inhibitor. Preferably, the compound is not Tenascin-C, a lysyl oxidase inhibitor, or ChABC. Inhibitors include polypeptides, small molecules (e.g., β-am nopropionitrile), and nucleic acid based inhibitors. Preferably, the inhibitor of a protein is a nucleic acid molecule such as an antisense oligonucleotide, an RNA

interference molecule or a binding molecule (e.g., an antibody or antibody fragment) that binds to the protein interferes with its function. A preferred inhibitor of hyaluronic acid is hyaluronidase. A preferred inhibitor of CD44 is an anti-CD44 antibody or antigen binding fragment thereof. Preferably the compounds are nucleic acids encoding polypeptides in which the polypeptide is membrane anchored. Preferably, said protein is selected from chondroitinase ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue-type plaminogen activator (tPA), and an antibody, preferably an antigen binding fragment, that bind sulphation motifs of CSPGs. The addition of a membrane anchor is thought to limit the spread of the expressed protein so that the effects are localized to the hippocampus. Preferably, said nucleic acids also comprise a hippocampal specific promoter.

Accordingly, the disclosure further provides an isolated nucleic acid encoding a recombinant protein, which is membrane anchored upon expression in a cell, wherein said recombinant protein alters the amount or the composition of the extracellular matrix (ECM) in the hippocampus. Preferably the protein and the membrane anchor are from different proteins, i.e., a chimeric protein is expressed.

In some embodiments the compound contains an endogenous membrane anchor (e.g., GPI-linked MMP17, type I transmembrane MMP14, and type II transmembrane MMP23). In other embodiments, the compound is a chimeric protein comprising an exogenous membrane anchor, e.g., the type I

transmembrane MMP14 fused to neurotrypsin. Suitable means to anchor a polypeptide to the membrane are known in the art and include the transmembrane domain from a membrane protein (e.g., the transmembrane region of the HLA class I or CD4 proteins) as well as GPI- anchors. In some embodiments, the polypeptide comprises a GPI-signal peptide. Such a signal peptide is a C-terminal amino acid sequence of a polypeptide which consists of one amino acid to which the GPI-anchor will be attached, an optional spacer peptide, and a hydrophobic peptide. Almost all of this signal peptide, i.e. the optional spacer peptide and the hydrophobic peptide, is removed posttranslationally by the enzyme GPI- transaminase and a bond between the amino group of the core ethanolamine phosphate of the GPI-anchor and the amino acid to which the GPI-anchor is attached is formed. Methods for preparing such recombinant proteins are known in the art and additional details may be found in WO2007131774, which is hereby

incorporated by reference.

Preferably, the nucleic acids encoding polypeptides as disclosed herein also contain a signal sequence directing the polypeptide to the cell membrane, usually a N-terminal stretch of around 20 amino acids

The compounds may be provided as isolated nucleic acids. As used herein, "isolated" means that the polypeptides are separated from other components of either (a) a natural source, such as a plant or cell, preferably bacterial culture, or (b) a synthetic organic chemical reaction mixture. Said nucleic acids may be operably hnked to additional sequences such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In a preferred embodiment, the nucleic acid is operably linked to a hippocampal promoter. Such promoters are described, e.g., in Valen et al. Genome Res. 2009 Feb; 19(2):255-65 and the Dlx5/6 promoter described in Delzor et al. Human Gene Therapy Methods 2012 23:242-254

In some embodiments, the compound is an inhibitor of a protein. Preferably the inhibitor is a nucleic acid molecule whose presence in a cell causes the degradation of or inhibits the function, transcription, or translation of its target gene, e.g., Bean, in a sequence-specific manner. Exemplary nucleic acid molecules include aptamers, siRNA, artificial microRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense oligonucleotides, and DNA expression cassettes encoding said nucleic acid molecules. Preferably, the nucleic acid molecule is an antisense oligonucleotide. Antisense oligonucleotides (ASO) generally inhibit their target by binding target mRNA and sterically blocking expression by obstructing the ribosome. ASOs can also be used for "exon-skipping". Exon-skipping oligonucleotides bind to pre-mRNA and modulate splicing such that one or more exons are skipped in the resulting mRNA. Exon-skipping may lead to an in frame deletion resulting in a truncated protein or protein lacking internal amino acids or skipping may lead to a premature stop codon resulting in nonsense-mediated decay. The design of such oligonucleotides is well-known in the art (see, e.g., Aartsma-Rus et al Mol Ther 17(3):548 (2009)).

ASOs can also inhibit their target by binding target mRNA thus forming a DNA-RNA hybrid that can be a substance for RNase H. ASOs may also be produced as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Methods for designing and modifying such gapmers are described in, for example, U.S. Patent Publication Nos. 20110092572 and 20100234451. Preferred ASOs include Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), and morpholinos.

Preferably, the nucleic acid molecule is an RNAi molecule, i.e., RNA

interference molecule. Preferred RNAi molecules include siRNA, shRNA, and artificial miRNA. siRNA comprises a double stranded structure typically containing 15 to 50 base pairs and preferably 19 to 25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. As used herein "shRNA" or "small hairpin RNA" (also called stem loop) is a type of siRNA. In one embodiment, these shRNAs are composed of a short, e.g. about 10 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow.

The design and production of siRNA molecules is well known to one of skill in the art (Hajeri PB, Singh SK. Drug Discov Today. 2009 14(17-18):851-8).

Methods of administration of therapeutic siRNA is also well-known to one of skill in the art (Manjunath N, and Dykxhoorn DM. Discov Med. 2010

May;9(48):418-30; Guo J et al, Mol Biosyst. 2010 Jul 15;6(7): 1143-61). siRNA molecule comprises an antisense strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence or a portion thereof encoding. Artificial miRNA molecules are pre-miRNA or pri-miRNA comprising a stem- loop structure(s) derived from a specific endogenous miRNA in which the stem(s) of the stem -loop structure(s) incorporates a mature strand-star strand duplex where the mature strand sequence is distinct from the endogenous mature strand sequence of the specific referenced endogenous miRNA. (See, e.g., U.S. Patent Publication Nos. 20050075492 and 20100292310 for the design and production of artificial miRNA molecules).

RNA interference refers to a decrease in the mRNA level in a cell for a heterologous target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the, e.g., miRNA or siRNA interference molecule RNAi molecules may also include chemical analogues such as, e.g., 2'-0-Methyl ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and other chemical analogues of nucleic acid oligonucleotides.

The nucleic acid molecule inhibitors may be chemically synthesized and provided directly to cells of interest. The nucleic acid compound may be provided to a cell as part of a gene delivery vehicle. Such a vehicle is preferably a liposome or a viral gene delivery vehicle. Liposomes are well known in the art and many variants are available for gene transfer purposes. Vectors comprising said nucleic acids are also provided. A "vector" is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include retrovirus, adeno-associated virus (AAV), pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors. Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.). Lentiviruses have been previously described for transgene delivery to the hippocampus (van Hooijdonk BMC Neuroscience 2009, 10:2) There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993)). Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided. The method of introduction is largely dictated by the targeted cell type include, e.g., CaP0 ₄ precipitation, liposome fusion, lipofectin,

electroporation, dextran-mediated transfection, calcium phosphate

precipitation, polybrene mediated transfection, protoplast fusion, , viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. The nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). Such cells are useful for producing isolated polypeptides which may be used in the methods described herein.

The compounds as described herein may be provided as isolated polypeptides. Preferably, via conventional techniques, the polypeptides are purified.

Polypeptides as described herein may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a dominant negative polypeptide. Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtihs, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc.

Preferably, said polypeptides are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. Suitable cell types include tumor cells, Jurkat T cells, NIH3T3 cells, CHO, and Cos, cells.

In some embodiments, the inhibitors described herein are antibodies, e.g., antibodies that block the sulfation motifs of CSPGs. As used herein, the term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric

antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Methods of making antibodies are well known in the art and many suitable antibodies are commercially available. Preferably, the antibodies disclosed herein include antigen binding fragments (e.g., Fab', F(ab')2, Fv or Fab fragments). The disclosure further provides compositions comprising a compound that alters the amount of or the composition of the extracellular matrix in the hippocampus and a pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided. Such

pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer, diluent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. When administering the pharmaceutical preparations thereof to an individual, it is preferred that the compound is dissolved in a solution that is compatible with the delivery method. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Preferred are excipients capable of forming complexes, vesicles and/or liposomes that deliver such a compound as defined herein in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients comprise polyethylenimine (PEI) or similar cationic polymers, including polypropyleneimine or

polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT- 18), lipofectin™, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, to a cell.

In a preferred embodiment, a compound is provided directly to the

hippocampus. The compound may be delivered by way of a catheter or other delivery device having one end implanted in a tissue, e.g., the brain by, for example, intracranial infusion. Such methods are known in the art and are further described in U.S. Publications 20120116360 and 20120209110, which are hereby incorporated by reference. A compound as described herein may also be administered into the cerebral spinal fluid. Such compounds are preferably linked to molecules that preferentially bind hippocampal cells (e.g., molecules that bind hippocampal specific cell surface molecules).

Methods that use a catheter to deliver a therapeutic agent to the brain generally involve inserting the catheter into the brain and delivering the composition to the desired location. To accurately place the catheter and avoid unintended injury to the brain, surgeons typically use stereotactic

apparatus/procedures, (see, e.g., U.S. Pat. No. 4,350, 159) During a typical implantation procedure, an incision may be made in the scalp to expose the patient's skull. After forming a burr hole through the skull, the catheter may be inserted into the brain. Other delivery devices useful with methods disclosed herein include a device providing an access port, which can be implanted subcutaneously on the cranium through which therapeutic agents may be delivered to the brain, such as the model 8506 ICV Access Port and the 8507 Intraspinal Port, developed by Medtronic, Inc. of Minneapolis, Minn.

Actual dosage levels of the pharmaceutical preparations described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical

composition required. For example, the physician or veterinarian could start with doses of the compounds described herein at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The disclosure also contemplates the treatment of individuals having or being at risk of developing age-dependent cognitive decline and/or memory

impairment, or a neurodegenerative disorder in which age-dependent cognitive decline and/or memory impairment are accelerated, comprising conjointly administering to an individual in need thereof one or more compounds that alter the amount of or the composition of the extracellular matrix (ECM) in the hippocampus of said individual and a composition for the treatment of cognitive decline or memory impairment. Said compositions may be

administered simultaneously or sequentially. A preferred composition for the treatment of cognitive decline or memory impairment comprises a cholinesterase inhibitor (ChEIs). These include;

tacrine, donepezil, rivastigmine and galantamine. The administration of such compounds is described in U.S. Publication No. 20060160079, which is hereby incorporated by reference. Compositions may also include inflammatory mediaters as described in U.S. Publication No. 20110142795 or memantine.

The disclosure further provides biomarkers and diagnostic methods for determining the risk of an individual developing age-dependent cognitive decline and/or memory impairment, or a dementia. The methods provided herein are especially well-suited for the early detection of said disorders. Preferably, said method is for determining the risk of developing Alzheimer's disease in an individual in Braak stages 1-3. Such individuals exhibit only very mild or no symptoms of cognitive decline. Preferably said individual is in Braak stages 1-2, more preferably in stage 1.

In a further aspect, the disclosure provides a method of classifying an individual as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia, comprising determining the level of one or more extracellular matrix (ECM) components in the hippocampus of said individual. An increased level of ECM components as compared to a reference indicates that said individual has or is at risk of developing a cognitive, memory, or dementia.

Preferably, said ECM component is selected from perlecan, laminin, fibronectin, collagen, tenascins, such as Tenascin-C (Tnc) and Tenascin-R

(Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl); thrombospondin, heparin-sulfate proteoglycan agarin, chondroitin sulphate proteoglycans (preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan); chondroitin sulfate; heparin; glycosaminoglycan, and sulfated glycosarninoglycan. More preferably, said ECM component is selected from Versican and CD44.

Preferably, said ECM component is not Tenasin C. In preferred embodiments, the method comprises determining the level of two or more extracellular matrix (ECM) components in the hippocampus of said individual. Preferably, said method comprises determining the level of Tenasin C and Versican or Tenasin C and CD44. In a preferred embodiment of the disclosure, the level of one or more ECM components in the hippocampus is determined by detecting one or more ECM components in the hippocampus, preferably using non-invasive detection. In preferred embodiments, the components in the hippocampus may be detected using an extracellular matrix binding compound (ECM-binding compound). ECM-binding compounds suitable for use include compounds that bind, e.g., to perlecan, laminin, fibronectin, collagen, xylosytransferase-1, tenascins, such as Tenascin-C (Tnc) and Tenascin-R (Tnr); pentraxins, pleiotrophin/HB-GAM, reelin, hyaluronan link protein (e.g., Haplnl);

thrombospondin, heparin-sulfate proteoglycan agarin, chondroitin sulphate proteoglycans (preferably a CSPG selected from aggrecan, brevican (Bean), neurocan (Ncan), NG2, versican (Vcan) and phosphacan); chondroitin sulfate; heparin; glycosaminoglycan, and sulfated glycosarninoglycan.

In some embodiments, the ECM-binding compound is a small molecule, a peptide, a protein, or an antibody. The term "antibody" includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments.

In some embodiments, the ECM-binding compound is a hyaluronan binding protein. Suitable proteins or binding fragments thereof include a CD44 polypeptide, a TSG6 polypeptide, an HABP4 polypeptide, an HAPLN1 polypeptide, an RHAMM polypeptide, a STAB-1 polypeptide, a STAB-2 polypeptide,; an XLKD1 polypeptide, a brevican polypeptide, an LYVE-1 polypeptide, an aggrecan polypeptide a versican polypeptide, a neurocan polypeptide. In some embodiments, the ECM-binding compound is Wisteria floribunda agglutin (WFA), which labels chondroitin sulfates.

Accordingly, the disclosure provides methods as described herein of classifying an individual as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia, comprising determining the level of one or more extracellular matrix (ECM) components in the hippocampus of said individual, further comprising providing to said

individual an ECM-binding compound, and determining the amount of ECM- binding compound in the hippocampus of said individual.

Preferably, said ECM-binding compound further comprises a label allowing for its detection. In preferred embodiments, positron emission tomography is used to determine the level of one or more ECM components. Accordingly, a method is provided comprising providing an individual with a PET compatible tracer, wherein said tracer binds one or more ECM components, and scanning said individual with a PET scanner.

PET is a well-known technique to determine the distribution of a tracer in vivo. A radioactive tracer is administered to an individual. The individual is then subjected to a scanning procedure using a PET or PET/CT scanner.

Quantification of radiopharmaceutical (radio-tracer) uptake by the target tissue can be performed using methods known in the art (see, e.g., Boellaard R. et al. Journal of Nuclear Medicine, Vol. 45, No. 9, pp 1519-1527, 2004 and U.S. Publications 20100196274 and 20110148861, which are hereby incorporated by reference). The distribution of ECM binding tracer can be determined in

"normal" individuals to determine a baseline which can be compared to the level in subject suspected of cognitive/memory impairment. A suitable tracer binds to said one or more ECM components and is preferably a peptide sequence. The tracer is labelled with a short-lived radioactive tracer isotope, such as carbon-11, nitrogen-13, oxygen-15, or fluorine-18.

In other preferred embodiments of the disclosure, the level of one or more ECM components in the hippocampus is determined by detecting one or more ECM components in a biological sample from said individual, preferably from blood, serum, or cerebrospinal fluid, more preferably from blood or serum.

As is understood by a skilled person, the detection of ECM components includes and is preferably the detection of peptide fragments of ECM proteins. Peptide fragments are understood as being from 10 to 100 amino acids in length, preferably from 10 to 50 amino acids in length.

An ECM component can be detected using a number of assays known to a skilled person. Preferred assays are based on antibody binding to ECM component, in particular peptide fragments of ECM proteins. Commercially available antibodies exist for the detection of ECM proteins and additional antibodies can easily be prepared by methods known in the art. Suitable immunoassays include, e.g., radio-immunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein A assay, and Immunoelectrophoresis assay. In addition to the use of antibody based assays, assays using other ECM component binders may also be used. For example, an ECM binding peptide can be immobilized on a solid support such as a chip. A biological sample is passed over the solid support. Bound ECM components are then detected using any suitable method, such as surface plasmon resonance (SPR) (See e.g., WO 90/05305, herein incorporated by reference).

Preferably, the method comprises the detection of Versican or a Versican peptide with a Versican specific antibody. Preferably, the method comprises the detection of CD44 or a CD44 peptide with a CD44 specific antibody.

Preferably, the method comprises the detection of Tenascin C or a Tenascin C peptide with a Tenascin C specific antibody and the detection of

CD44 or a CD44 peptide with a CD44 specific antibody and/or the detection of Versican or a Versican peptide with a Versican specific antibody.

Preferably, an individual is classified as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or a dementia when there is a significant increase in one or more ECM components in a biological sample from said individual as compared to a reference value.

A "significant" increase in a value, as used herein, can refer to a difference which is reproducible or statistically significant, as determined using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. Preferably, a significant increase is at least 20, at least 40, or at least 50% higher than the reference value.

A reference value refers to the level (amount) of a protein in a comparable sample (e.g., from the same type of tissue as the tested tissue, such as blood or serum), from a "normal" healthy subject that does not exhibit cognitive decline and/or memory impairment and/or a neurodegenerative disorder. If desired, a pool or population of the same tissues from normal subjects can be used, and the reference value can be an average or mean of the measurements. Definitions

As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

The word "approximately" or "about" when used in association with a

numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

The term "treating" includes prophylactic and/or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the

invention. EXAMPLES

EXPERIMENTAL PROCEDURES

Animals

All animals in this study were C57BL/6J mice. C57B1/6J breeders were received from Charles River (Lyon, France) and were bred in the animal facility of the VU University Amsterdam. All animal experiments were approved by the Animal Welfare Committee (DEC) of the VU University (protocol MCN 09-12).

APP-PS1 mutant mice

For details see The Jackson Laboratory [strain B6C3-Tg(APPswe, PSEN1- dE9)85Dbo/J; stock number 004462; http://jaxmice.jax.org/]. APP-PSl transgenic mice express a chimeric mouse/human APP gene harboring the Swedish double mutation K595N/M596L (APPswe) and a human PSl gene harboring the exon 9 deletion (PSldE9), both under the control of the mouse prion protein promoter (MoPrP.Xho) (Jankowsky, Slunt et al. 2001;

Jankowsky, Xu et al. 2003; Jankowsky, Fadale et al. 2004). AD mice were maintained as hemizygous and crossed with WT C57BL/6. WT littermates served as controls for both AD mouse lines. Synaptosome isolation and sample preparation

Hippocampi were dissected, frozen, and stored at -80°C until protein isolation. Synaptosomes were isolated from hippocampi at eight different ages, 20 weeks, 40 weeks, 50 weeks, 60 weeks, 70 weeks, 80 weeks, 90 weeks and 100 weeks, as described previously (Li, Miller et al. 2007; Klychnikov, Li et al. 2010) with minor modifications. In brief, hippocampi were homogenized in ice-cold 0.32 M sucrose buffer with 5 mM HEPES at pH 7.4 and protease inhibitor (Roche) and centrifuged at 1000 x g for 10 min at 4°C to remove debris. Supernatant was loaded on top of a discontinuous sucrose gradient consisting of 0.85 M and 1.2 M sucrose. After ultracentrifugation at 110,000 x g for 2 h at 4°C, the synaptosome fraction was collected at the interface of 0.85 M and 1.2 M sucrose, resuspended and pelleted by ultracentrifugation at 80,000 x g for 30 min at 4°C. After which the material was redissolved in 5 mM HEPES. Protein concentrations were determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). For each sample, 50 g of protein was transferred to a fresh tube and dried in a SpeedVac overnight.

Protein digestion and iTRAQ labeling

Per sample, 50 g synaptosome proteins were dissolved in 0.85% RapiGest (Waters Associates, Milford, MA, USA), alkylated with methyl

methanethiosulfonate, and digested with trypsin (sequencing grade; Promega, Madison, WI, USA) as described (Li, Miller et al. 2007; Van den Oever, Goriounova et al. 2008). In each iTRAQ experiment samples were labeled respectively with iTRAQ reagents 113 = 20 weeks, 114 = 40 weeks, 115 = 50 weeks, 116 = 60 weeks, 117 = 70 weeks, 118 = 80 weeks, 119 = 90 weeks, and 121 = 100 weeks. To accommodate eight independent protein samples for each time point, a total of eight 8-plex iTRAQ experiments was performed (8x8).

Two-dimensional liquid chromatography (2DLC)

The lyophilized iTRAQ labeled samples were separated in the first dimension by strong cation exchange column (2.1x150 mm polysulfoethyl A column,

PolyLC), and the in second dimension on an analytical capillary reverse phase C18 column (150 mm x 100 μιη i.d. column) at 400 nL/min using the LC- Packing Ultimate system. The peptides were separated using a hnear increase in concentration of acetonitrile from 4 to 28% in 75 minutes, to 36% in 7 minutes and finally to 72% in 2 minutes. The eluent was mixed with matrix (7 mg of re-crystallized a-cyano-hydroxycinnaminic acid in 1 ml 50%

acetronitrile, 0.1% trifluoroacetic acid, 10 mM ammonium dicitrate) and delivered at a flow rate of 1.5 μΐνηιίη and deposited onto an Applied

Biosystems matrix-assisted laser desorption ionization plated by means of a robot (Probot, Dionex) once every 15 sec for a total of 384 spots.

MALDI-MS/MS

Samples were analyzed on an ABI 5800 proteomics analyzer (Applied

Biosystems, Forster City, CA). Peptide collision -induced dissociation was performed at 2 kV, the collision gas was air. MS/MS spectra were collected from 2000 laser shots. Peptides with signal-to-noise ratios over 50 at the MS mode were selected for MS/MS analysis, at a maximum of 30 MS/MS per spot. The precursor mass window was set to a relative resolution of 200. Peaklists were extracted from the instrument database using TS2Mascot software (MatrixScience).

Protein identification

Protein identification and quantification are described in detail in (van Nierop and Loos 2011). To annotate spectra, Mascot (MatrixScience, version 2.3.01) searches were performed against the Swissprot database (20/10/2010) and against the larger but more redundant NCBI database (20/10/2010). MS/MS spectra were searched with trypsin specificity and fixed iTRAQ modifications on lysine residues and N-termini of the peptides and methylthio modifications on cysteine residues. Oxidation on methionine residues was allowed as a variable modification. Mass tolerance was 150 ppm for precursor ions and

0.5 Da for fragment ions, while allowing a single site of miscleavage. The false discovery rate (FDR) for peptides identification was calculated using a randomized database. Protein redundancy in the result files was removed by clustering the precursor protein sequences at a threshold of 90% sequence similarity over 85% of the sequence length. Subsequently all peptides were matched against the protein clusters and only those peptides were included that mapped unique to one protein. Proteins were considered for quantification if at least two unique peptides had a confidence interval of > 95%, and at least three peptides were identified in four replicate iTRAQ sets and at least one peptide in the other sets.

Protein quantification and identification of differentially expressed proteins in aging study

iTRAQ areas (m/z 113-121) were extracted from raw spectra and corrected for isotopic overlap using GPS explorer. Peptides with iTRAQ signature peaks of less than 750 were not considered for quantification. To compensate for the possible variations in the starting amounts of the samples, the individual peak areas of each iTRAQ signature peak were log transformed to yield a normal distribution, and normalized to the mean peak area of every sample. Protein abundances in every experiment were determined by taking the average normalized standardized iTRAQ peak area of all unique peptides annotated to a protein. Finally, the standardized protein means were used to calculate the average abundance at each time point relative to week 20. To determine which proteins show significant differential expression, three types of statistical analysis were performed. First, the One-Class Time Course Analysis option implemented in the Significance Analysis of Microarrays (SAM) package (Tusher, Tibshirani et al. 2001) was used to determine significant regulation over time. In addition, the Two-Class Analysis option in SAM was used to determine significant regulation at individual time points. Two-class SAM analysis was used both as an unpaired analysis, and as a paired analysis in which the 8 samples within iTRAQ sets were considered as paired with the 20 weeks time point as the base line. Paired analysis was performed to reduce false positives resulting from technical variation between iTRAQ sets, and resulted in more stringent selection of differentially regulated proteins than unpaired analysis. In all SAM analyses a threshold q-value of 10% was used to determine significant differential protein expression. The Time Course

Analysis was used to determine the set of proteins for which there was strongest evidence for regulation over time. K-means clustering and Pearson correlation analysis were used to further analyze this set of proteins. The intersection of the paired and the unpaired Two-Class Analysis was used to determine the set of proteins for which there was evidence for regulation at one or more time points.

Functional protein group analysis

All proteins were assigned to one of 17 functional synaptic protein groups as previously defined (Lips, Cornelisse et al. ; Ruano, Abecasis et al. 2010), and overrepresentation of regulated proteins within functional groups was determined using a Fisher's exact test. Enrichment was only considered relevant when overrepresented functional groups contained at least 3 proteins. In addition, functional enrichment was determined using the DAVID

functional annotation tool (http://david.abcc.ncifcrf.gov/) (Huang da, Sherman et al. 2009; Huang da, Sherman et al. 2009). The functional categories used were GO term related to Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), as well as pathway annotations derived from KEGG. The entire set of detected proteins was used as the background set, and a Benjamini FDR <0.1 was considered significant. Enrichment was only considered relevant when enriched functional groups contained at least 5 proteins or at least 3 proteins in the AD study. Protein variance analysis

For each detected protein, mean standardized peak areas and standard deviations (SD) of the mean standardized peak areas were calculated per time point for both the original dataset and subtracted data set (paired against the 20 week time point). Parametric one-way ANOVA and non-parametric

Kruskal-Wallis (Siegel and Castellan 1988) tests were used to determine whether the distribution of these sample means and SDs are dependent on time. Next, all SDs from all time points were collected and ranked, and the top 5% was selected and designated high variable proteins, whereas the bottom 5% was selected and designated low variable proteins. The intersection of the original and subtracted data was used to determine the set of proteins with either high or low variability. The distribution of high and low variable proteins over time points was determined, and functional enrichment analysis was performed using DAVID (http://david.abcc.ncifcrf.gov/) (Huang da, Sherman et al. 2009; Huang da, Sherman et al. 2009), using as a background the whole set of detected proteins and a significance threshold of Benjamini FDR <0.1.

Immunob lotting

Western blot analysis was performed on six synaptosome protein extracts (six biological replicates) per time point. For detection of extracellular matrix proteins, samples were treated with chondroitinase ABC (Sigma Aldrich, Zijndrecht, The Netherlands; 0.5 U/50 mg protein) for 90 min at 37°C. Of each sample, 2.5 μg protein was mixed with SDS sample buffer and heated to 90°C for 5 min. Proteins were separated on a Criterion™ TGX Stain-Free Precast Gel (4-16% Tris-Glycine; Bio-Rad) in a Criterion™ Cell Electrophoresis System (Bio-Rad), and electroblotted onto PVDF membrane overnight at 4°C. After blocking with 5% non-fat dry milk in TBS-T (TBS plus 0.5% Tween) for 1 h, blots were incubated with primary antibodies, followed by a horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark;

1: 10,000). The following antibodies were used: anti-Brevican (gift from Dr. C. Seidenbecher, Magdeburg, Germany; 1:2,000), anti-Haplnl (Abeam; 1: 1,000), anti-Neurocan (Sigma; 1: 1000). Blots were incubated with ECL substrate (GE Healthcare, Pollards Wood, UK), scanned with a Odyssey Imager (LI-COR) and analyzed with Image Studio software (LI-COR, version 1.1.7) using background correction. To correct for differences in sample input, Criterion™ TGX Stain-Free Precast Gel after SDS-page and PVDF membrane after electroblotting were visualized, and the densitometric values were used for normalization (Van den Oever, Goriounova et al. 2008; Counotte, Li et al. 2010). Significance was determined using a Student's i-test (one-tailed, independent samples).

Immunohistochemistry

Immunohistochemistry was performed on brain sections at 3 months (P90), 6 months (P180), 9 months (P270), and 1 year (P365) of age. Brains were fixed by transcardial perfusion with 4% paraformaldehyde in phosphate buffered saline (n=4-6 pairs). Coronal cryosections (10 m) were thaw-mounted on Superfrost Plus slides, dried for 1 h at room temperature, and stored at -20°C until use. Sections were fixed with fresh 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.0, for 10 min at room temperature. Sections were treated using 10 mM sodium citrate and 0.05% Tween 20 (pH 6.0) for 20 minutes at 95°C, blocked 10% normal donkey serum and 0.4% Triton X-100 in PBS, and were incubated overnight with 6E10 (Signet;

1: 15000) and GFAP (DAKO; 1:2000). Antigens were visualized using Cy3 and DyLight 488 (Jackson ImmunoResearch Laboratories and Invitrogen; 1:400) incubated for 2 h at room temperature. Sections were washed and coverslipped in Vectashield including DAPI as a nuclear dye (Vector Laboratories). WFA and Tnr immunohistochemistry were performed on free-floating brain sections obtained from animals at 3 months of age. Sections were quenched (10% methanol, 0.3% H ₂ 0 ₂ in PBS) for 30 minutes, blocked with 0.2% Triton X-100 and 5% fetal bovine serum in PBS, and were incubated overnight with fluorescent labeled WFA (Vector Laboratories; 1:400) and Tnr (P.Glia, Bonn, Germany; 1:400). Tnr was visualized using DyLight 488 (Invitrogen; 1:400) incubated for 2 h at room temperature. Sections were washed and coverslipped in Vectashield including DAPI as a nuclear dye (Vector Laboratories). Quantification of differentially expressed proteins in AD study

iTRAQ peak areas (m/z 113-121) were extracted from raw spectra and corrected for isotopic overlap using GPS explorer. Peptides with iTRAQ signature peaks of less than 750 were not considered for quantification. To compensate for small differences in the starting amounts of the samples, the individual peak areas of each iTRAQ signature peak were log transformed to yield a normal distribution, and normalized to the mean peak area per sample. Protein abundance was determined by taking the average normalized standardized iTRAQ peak area of all unique peptides annotated to that protein. Finally, the standardized protein means (four mutant and four wildtype in each experiment) were used to calculate the average log-fold difference between WT and AD mice. To assess whether differences had occurred by chance or could be deemed significant, we calculated permutation- derived false discovery rates (FDR), using Significance Analysis of Microarrays (SAM) (Tusher, Tibshirani et al. 2001) as implemented in the Multi

Experiment Viewer software (MeV, version 4.6.1) (Tusher, Tibshirani et al. 2001; Saeed, Bhagabati et al. 2006). SAM uses a data resampling-based method and creates randomized data distributions in order to estimate false positive rates (Tusher, Tibshirani et al. 2001), and can be successfully applied to proteomics data (Roxas and Li 2008; Counotte, Li et al. 2010; Mueller, Denef et al. 2010; Dahlhaus, Wan Li et al. 2011; Klemmer, Meredith et al. 2011). Conventional i-test or fold change alone do not take into account the effect of multiple testing, and SAM is more adaptive to different biological experiments that may have asymmetrical distribution of differential protein expression profiles. The SAM q-values reflect for each protein the number of empirically determined false-positives at the significance level of the respective protein. The false discovery rate (FDR) levels in our results thus hold information about individual proteins, and should not be interpreted as a global FDR.

Changes in protein expression are considered to be significant when the FDR is <10% and log-fold change >0.125. In addition, we have also provided the corresponding p-value as determined by Student's i-test.

Contextual fear conditioning

All experiments were carried out in a fear conditioning system (TSE Systems) as previously described (Rao-Ruiz, Rotaru et al. 2011). Training and testing was performed in a Plexiglas chamber with a stainless steel grid floor with constant illumination (100-500 lx) and background sound (white noise, 68 dB sound pressure level), situated in a gray box to shield it from the outside. The chamber was cleaned with 70% ethanol before each session. Training consisted of placing mice in the chamber for a period of 180 s, after which a 2 s foot shock (0.7 mA) was delivered through the grid floor. Mice were returned to their home cage 30 s after the end of the shock. The retrieval tests consisted of a 3 min re-exposure to the context (conditioned stimulus) 24 h after

acquisition. Baseline inactivity, exploration, distance traveled and freezing were assessed automatically. Freezing was defined as lack of any movement besides respiration and heart beat during 4 s intervals and is presented as a percentage of the total test time. APP-PSl transgenic mice (n=l l) and wildtype littermate controls (n=l l) were tested at 3 months of age.

Significance was determined using a one-tailed Student i-test.

RESULTS

Proteomic analysis identifies 25 proteins that are significantly regulated over time

To identify and quantify age-related changes in synaptic protein expression, we performed 8-plex iTRAQ proteomics of hippocampal synaptosomes isolated from C57BL/6 mice at 20, 40, 50, 60, 70, 80, 90 and 100 weeks of age. For each time point, eight independent biological replicate samples were measured. Each iTRAQ experiment contained samples from all eight time points, allowing comparison within and between age groups (Figure 1). In total, 502 high confidence proteins were detected (CI >95%; number of quantifiable peptides >3 in at least 4 experiments, and >1 in four other experiments;

Supplemental Table Si). In the text we will refer to proteins as upregulated' or 'downregulated', when their expression level is higher or lower respectively than at the 20 week age. To select differentially expressed proteins that are consistently up- or downregulated over time, we used the one-class time course analysis option in the Significance Analysis of Microarrays (SAM) package (Tusher, Tibshirani et al. 2001). Time course analysis revealed a total of 17 proteins significantly upregulated over time, and 8 proteins significantly downregulated over time (Figure 2A). K-means clustering was used to separate these 25 proteins into 4 different expression clusters (Figure 2 A). Clusters 1-3 contained proteins whose expression was increased or decreased already at early time points (40-50 weeks), and remained relatively constant over time thereafter. Only proteins in cluster 4 showed progressive upregulation over time until week 90. These proteins also showed the strongest correlation between expression and time, as evidenced by R correlation coefficients (Figure 2A). Interestingly, three out of the four proteins in this cluster represent protein constituents of the extracellular matrix (ECM; i.e., Haplnl, Ncan and Bean) (Figure 2B). Western blotting confirmed the age-dependent upregulation of each of these three proteins (Figure 2C). Together, these findings suggest that the most significant and characteristic age-dependent alteration in the hippocampal synaptic proteome is a progressive increase in synaptic ECM levels. Proteomic analysis identifies 101 proteins that are significantly regulated at any time point

Paired and unpaired two-class analyses in SAM were used to determine significant regulation at any time point. In the paired setup, the eight samples within each iTRAQ set were considered as paired, and were paired in respect to the 20 week age, which reduces false positives. The intersection of the paired and the unpaired analysis revealed 101 proteins being significantly differentially expressed at any time point (Figure 3A). To allow robust functional characterization of these proteins, and to reduce the impact of potential false negatives at individual time points, we first combined differentially expressed proteins into three different age groups: early-aged (weeks 40 and 50), middle-aged (weeks 60, 70 and 80), and old-aged (weeks 90 and 100). Whereas significantly upregulated proteins were present in all three age groups, a progressive increase was observed for significantly

downregulated proteins with age (Figure 3B). For functional analysis, all differentially expressed proteins were categorized in 17 functional synaptic protein groups as previously defined (Lips, Cornelisse et al. ; Ruano, Abecasis et al. 2010), and regulated proteins at each age group were separately tested for overrepresentation of functional protein groups using a Fisher's exact test (Figure 3C; Table 1). In early-aged mice, we observed overrepresentation of upregulated proteins involved in cell metabolism and structural plasticity (Figure 3C-D). In middle-aged mice, overrepresentation was observed for proteins involved in cell adhesion (Figure 3C-D). There was no significant overrepresentation found for any functional group in old-aged mice.

As a complementary approach, functional enrichment of differentially expressed proteins was determined using gene ontology (GO) and cellular pathway (KEGG) databases. Enrichment was determined separately for each age group (early-, middle-, and old-aged). Table 2 lists all overrepresented GO terms and KEGG pathways per age group, as well as the main direction of regulation for the proteins observed in each of these GO classes and KEGG pathways. Early-aged mice show an upregulation of several GO classes related to cytoskeletal proteins, in particular tubulins (i.e., Tubb3, Tubb4a, Tubb5, Tppp), and ECM (i.e., Ncan, Bean, Haplnl). Middle-aged mice show both up- and downregulation of GO classes related to the cortical cytoskeleton (e.g., Acnt4, Nefl, Nefm), non-membrane bound organelles (e.g., Kif5c, Rps4x), and microtubule-based processes and movement (e.g., Tubb4, Tubb5, Kif5c). The most prominent functional enrichment observed in old-aged mice involved an upregulation of several GO classes related to the ECM. Thus, in addition to confirming our previous analysis in showing a strong age-dependent

upregulation of the ECM, GO term and KEGG pathway overrepresentation analysis identified several other cellular functions that are affected by age.

Variance in synaptic protein expression increases with age

Increased variance in gene or protein expression might provide a possible causative explanation for neurodegenerative disorders (Mar, Matigian et al _¬ 2011). We therefore measured stochasticity in synaptic protein expression as a possible cause for age-related synaptic dysfunction and cognitive decline. We calculated the standard deviations (SD) of the average standardized peak areas per protein per time point, and tested whether SD distribution changes with age. We observed a significant increase in SD variance with age (original data: one-way ANOVA test, p = 9.91xl0 ¹⁶ ; Kruskal-Wallis test, p = 2.2xl0 ¹⁶ ; Figure 4A), whereas the distribution of the average standardized peak areas did not change with age (original data: one-way ANOVA, p = 0.3828; Kruskal- Wallis, p = 0.006489; Figure 4B). Similar results were obtained with the subtracted dataset (data not shown); SD variance increased with age (one-way ANOVA test, p = 1.07" ¹¹ ; Kruskal-Wallis test, p = 9.99" ¹¹ ), whereas there was no change in the distribution of the average standardized peak areas (one-way ANOVA, p = 0.7639; Kruskal-Wallis, p = 0.1821). Next, we ranked all the SD values, and we selected the 5% proteins with the lowest SD values, which we designated low variabihty proteins, as well as the 5% proteins with the highest SD values, which we named high variability proteins (Figure 4C). The intersection of the original and subtracted data sets revealed 103 proteins with extreme SD values, either low variable or high variable, at different time points, (see, e.g. Figure 5).

We next selected the low variability and high variability proteins that are specifically associated with the two oldest age classes (weeks 90 and 100) and performed a GO term and KEGG pathway functional enrichment analysis (Figure 4D). Low variability proteins are specifically associated with

nucleocytoplasmic transport, establishment of protein localization, synaptic vesicle transport, cell junction, extracellular structure organization, and ATPase activity. High variability proteins, on the other hand, are involved in cellular respiration, and associated with Parkinson's disease and Huntington's disease pathways. Proteins within these disease pathways are mainly mitochondrial proteins, involved in mitochondrial complex I, NADH

dehydrogenase, and mitochondrial complex IV, cytochrome C oxidase, as well as mitochondrial membrane proteins, F-type ATPases, and ATP synthases. In addition, the neurodegeneration-related alpha-synuclein (Snca) protein was found to be highly variable.

Identification of changes in hippocampal synaptic protein composition in APP-PS1 mice

In order to identify age-dependent changes in the molecular composition of hippocampal synapses in APP-PSl mice we performed 8-plex iTRAQ

proteomics. Hippocampal synaptosomes were isolated from APP-PSl mice and their wildtype littermates at 1.5 months, 3 months, 6 months and 12 months of age. We choose these time points in order to be able to distinguish early synaptic changes (1.5 and 3 months) that precede A6 pathology, from late synaptic changes (6 and 12 months), which may be the consequence of Αβ pathology. The iTRAQ experiment was set-up as illustrated in Figure 3.1A, and included five independent biological replicates. A total of 376 proteins were identified (Figure 6). SAM analysis indicated that the levels of 104 proteins were significantly different between APP-PSl mice and wildtype controls at one or more time points (FDR <10, log-fold change >0.125; Figure 3. IB). Most differentially expressed proteins were detected at 3 months of age (86 proteins) compared to the other ages (0 proteins at 1.5 months, 1 at 6 months and 33 at 12 months; Figure 6C). Of these significantly regulated proteins, most proteins were downregulated, especially at 12 months of age, when almost all regulated proteins were downregulated (Figure 6C).

Surprisingly, classical synaptic proteins such as PSD95 (Dlg4), Bassoon (Bsn) and Piccolo (Pclo) were not significantly different between APP-PSl mice and wildtype controls, and also the major glutamate receptor subunits (GluRl,

GluR2, Grinl, and Grin2b) were not significantly different, although they were all detectable. This suggests that more subtle synaptic alterations may underlie cognitive deficits in APP-PSl mice. Increased expression of Αρρ/Αβ and ApoE in APP-PSl mice

The two most regulated proteins that were detected in hippocampal

synaptosomes were App and ApoE, which showed a log-fold increase of 1.32 and 0.74 respectively in APP-PSl mice compared with wildtype controls at 12 months of age. ApoE expression was only upregulated at 12 months of age (Figure 7A), which was confirmed by immunoblotting (Figure 7B). App expression was also significantly increased at 3 and 6 months of age (Figure 7C). Interestingly, higher App expression in AD mice at 3 and 6 months of age was observed for most App derived peptides, whereas the additional increase observed at 12 months was primarily due to an increase of the specific A6 peptide LVFFAEDVGSNK. The increase in A6 levels at 12 months was confirmed by immunoblotting using an anti-Ab 1-17 specific antibody (Figure 7D).

To correlate the observed increase in App/Ab expression with

pathological alterations in the brain, we next performed immunohistochemical stainings of hippocampal sections of APP-PSl mice at different ages using an antibody (6E10) specific for Ab. In accordance with earlier reports (Jankowsky, Fadale et al. 2004), no plaques were observed at 3 months of age. At 6 months, APP-PSl mice had few hippocampal plaques and plaque load steadily increased up to 12 months (Figure 7E). Parallel to the increase in plaque formation, there was a moderate increase in GFAP staining around all plaques at 12 months of age. Together, our data show that APP-PSl mice have increased levels of App expression and A6 accumulation in or around synapses. Importantly, this increase is already significant at 3 months, before any Αβ plaques are observed. The robust increase in ApoE expression observed at 12 months of age may reflect increased A6 clearance at hippocampal synaptic sites (Cramer, Cirrito et al. 2012).

Functional overrepresentation analysis of significantly regulated proteins

We next categorized all proteins in 17 functional synaptic protein groups as previously defined (Lips, Cornelisse et al. ; Ruano, Abecasis et al. 2010) (Figure 8A). Overrepresentation analysis was performed separately on regulated proteins at 3 and at 12 months of age. Enrichment of functional protein groups in these sets of regulated proteins was calculated relative to all detected proteins. At 3 months of age we observed an enrichment of proteins involved in cell adhesion/transsynaptic signaling, endocytosis, excitability, exocytosis, and G-protein relay (Figure 8A-B). At 12 months of age, enrichment was observed for proteins involved in cell adhesion/transsynaptic signaling, cell metabolism, exocytosis, and G-protein relay (Figure 8A-B). The expression profiles of individual proteins in these functional protein classes are depicted in Figure 8C. In addition, we also performed functional classification analysis based on GO annotation using DAVID. Using the Gene Functional Classification tool in DAVID, nine different functional groups were detected as significantly enriched in APP-PSl mice (Table 3.1). The highest enrichment scores were observed for proteins involved in cell adhesion, in particular the extracellular matrix (ECM). In addition, there was a significant enrichment for proteins involved in excitability, G-protein signaling, exocytosis, ion balance, and energy metabolism and mitochondrial respiration. Age-related changes in the extracellular matrix in APP-PS1 mice

Combined functional enrichment analysis using synaptic protein annotation and GO annotation indicated a highly significant upregulation of ECM proteins in APP-PSl mice compared to wildtype controls. Proteins in this group were neurocan (Ncan), tenascin-R (Tnr), hyaluronan and proteoglycan link protein 1 (Haplnl), and brevican (Bean). Comparison of the time course expression profiles of these proteins revealed a gradual age-dependent upregulation in wiltype mice, which was accelerated in APP-PSl mice, resulting in relatively higher expression levels at 3 and 6 months of age (Figure 9A). Immunoblotting confirmed the upregulation of all four ECM proteins in APP-PSl mice compared to wildtype controls at 3 months of age (Figure 9B). In addition, APP-PSl mice showed increased hippocampal staining using the ECM marker Wisteria floribunda agglutinin (WFA; Figure 10A). Specifically, we observed a strong increase in WFA-positive neurons in the subiculum and the adjacent tip of the CAl region of the hippocampus. This increase in WFA staining was observed in three independent APP-PSl mice compared with three independent wildtype controls (Figure 10B).

Reduced contextual fear memory in 3 months old APP-PSl mice

To investigate whether the early increase in synaptic ECM levels temporally correlate to early stages of memory decline in AD, we evaluated hippocampal spatial memory in APP-PSl transgenic mice and wildtype controls at 3 months of age using a contextual fear memory task. Training consisted of placing mice in a test chamber for a fixed period of time, after which a foot shock was delivered through the grid floor. Mice were returned to their home cage after the shock had ended. Retrieval tests consisted of re-exposure to the context (conditioned stimulus) at 24 h after acquisition (Figure 11A). We observed that wildtype and APP-PSl mice exhibited similar baseline activity during the training trials before shock delivery, as assessed by the percentage of inactivity and exploration and the distance moved (Figure 11B). However, memory performance, as assessed by freezing behavior on re-exposure of mice to the context, was reduced in APP-PSl mice compared with wildtype mice (Figure 11C). This effect almost reached statistical significance (p = 0.028). improved fear memory after treatment with an E CM modulating compound.

Three months-old APP-PSl animals and WT littermates (n=10 per genotype) were tested in a contextual fear memory paradigm (n=4 per genotype). APP- PSl animals showed a significant reduction in time spent freezing in the recall test (24h after conditioning), indicating impaired memory formation or recall (Figure 12). Next, a new group of three months-old APP-PSl animals and WT littermates (n=4 per genotype) received a cannula bilaterally into the dorsal hippocampus and were allowed to recover from the operation for 1 week. All animals then received a single dose of ChABC (0.025 U per side) and were conditioned 24h later. At recall (24h after conditioning), time spent freezing in APP-PSl animals was significantly higher than without ChABC treatment, and was at WT levels, indicating a rescue or contextual memory by local ChABC treatment in the hippocampus. Treatment with membrane anchored ECM modulating compound

Adeno-associated virus (AAV) encoding MMP 17, MMP 14, or MMP23 fused to GFP will be injected directly into hippocampus of mice. Locally injected AAV results in local expression of MMP and the endogenous membrane anchor will prevent further diffusion to other parts of the brain. Mice will be sacrificed and the lack of diffusion of GFP outside of the hippocampus will be observed. TABLE 1. Overrepresentation of functional protein classes per age- group. All quantified proteins were assigned to one of 17 functional synaptic protein groups. Significantly regulated proteins in each age-group (early-aged, middle-aged, old-aged) were separately tested for overrepresentation using a Fisher's exact test. Numerators represent the total number of proteins detected, denominators represent the number of proteins belonging to the indicated functional class.

Present in all Fisher's

Present in

Age group Functional group detected exact age-goup

proteins ju-value

Early- aged

Cell metabolism 503/33 30/8 0.0010

Structural plasticity 503/62 30/8 0.0315

Cell

Middle- adhesion/transsyna tic 22/6 0.0017 aged

signaling

TABLE 2. Functional enrichment of differentially expressed proteins.

Significantly enriched (Benjamini FDR <0.1) GO and KEGG terms are indicated per age group, as well as the main direction of regulation of the proteins within each GO or KEGG class.

Gene Ontology Young Middle Old

GO:0031344~regulation of cell projection organization UP

GO:0010975~regulation of neuron projection development UP

GO:0030182~neuron differentiation UP

GO:0030424~axon UP

GO:0030534~adult behavior UP

GO:0008344~adult locomotory behavior UP

mmu00270:Cysteine and methionine metabolism UP

GO:0005829~cvtosol UP UP/DOWN

GO:0034622~cellular macromolecular complex assembly UP

GO:0043933~macromolecular complex subunit organization UP

GO : 003462 l~cellular macromolecular complex subunit

organization UP

GO:0065003~macromolecular complex assembly UP

GO:0070271~protein complex biogenesis UP

GO:0043623~cellular protein complex assembly UP

GO: 000646 l~protein complex assembly UP

GO:0051258~protein polymerization UP

GO:0015630~microtubule cytoskeleton UP

GO:0000226~microtubule cytoskeleton organization UP

GO:0007017~microtubule-based process UP/DOWN GO:0007018~microtubule-based movement UP/DOWN

GO:0001871~pattern binding UP UP

GO:0030247~polysaccharide binding UP UP

GO:0005540~hyaluronic acid binding UP UP UP

GO:0005539~glycosaminoglycan binding UP UP Gene Ontology Young Middle Old

GO:0007155~cell adhesion UP GO:0022610~biological adhesion UP GO:0007010~c tos keleton organization UP UP/DOWN UP GO:0005576~extracellular region UP UP/DOWN GO:0031012~extracellular matrix UP UP/DOWN GO:0005578~proteinaceous extracellular matrix UP UP/DOWN UP GO:0044430~cytoskeletal part UP/DOWN GO:0030863~cortical cytoskeleton DOWN

GO:0030864~cortical actin cytoskeleton DOWN

GO:0043228~non-membrane-bounded organelle UP/DOWN GO:0043232~intracellular non-membrane-bounded

organelle UP/DOWN

GO:0006457~protein folding DOWN

GO:0016790~thiolester hydrolase activity DOWN mmu00480:Glutathione metabolism UP

mmu00980:Metabolism of xenobiotics by cytochrome P450 UP mmu00982:Drug metabolism UP

GO:0004364~glutathione transferase activity UP GO:0016765~transferase activity, transferring alkyl or aryl

UP

(other than methyl) groups

TABLE 3.1. Functional classification using DAVID. Enrichment was determined using the total set of all detected proteins as the background set, and using the following settings: Similarity Term Overlap: 4; Similarity Threshold: 0.35; Initial Group Membership: 4; Final Group Membership: 4; Multiple Linkage Threshold: 0.50.

Functional

class Group Gene name Enrichment score

Extracellular

matrix 1 Ncan, Bean, Haplnl, Cd200 0.815

Cell adhesion 2 Cd200, Ncaml, Icam5, Hnt 0.410

Cacng8, Kcnab2, Cacnb4,

Excitability 3 Cacna2d3 0.395

G-protein 4 Gnaq, Gnall, Gnaol, Gnaz 0.343

Rph3a, Syp, Stxlb, Syn2,

Exocytosis 5 Synl 0.343

Energy Ldha, Aldoc, Ldhb, Pkm2,

metabolism 6 Enol 0.302

Mitochondrion 7 Sdha, Idh3a, Suclgl, Sdhb 0.217

Atp6vlel, Atp6vlh, At lbl,

Ion balance 8 Atp6vla 0.063

Mitochondrion 9 Vdacl, Sfxnl, Samm50, 0.023

Cox4il, Phb Human Proteomics Study

A quantitative proteomics study was performed to map changes in the proteome of human post-mortem hippocampal regions (CAl and subiculum) at various stages of the disease.

Brain tissue selection was based on both the clinical and neuropathological diagnosis. Severity of dementia was measured using the Global Deterioration Scale (GDS; Reisberg, 1984), which encompasses activities of daily living, behavior and cognition. Each case was neuropathologically assessed and staged according to Braak and Braak (Braak and Braak, 1991). For each

Braak stage seven cases were selected (in total 49). The hippocampal subareas CAl and subiculum were isolated from each brain using lasercapture microdissection. Proteins were extracted from the brain tissue through lysis and separated by SDS-PAGE on the basis of their molecular mass. Proteins were in-gel digested, extracted and subsequently analyzed using mass spectrometry. In the data set -5000 proteins were identified, but as quantification requires multiple detection of proteins we could reliably quantify of ~ 2000 proteins over all seven Braak stages. Several groups of proteins are up- or down-regulated over the entire Braak series, which may be indicative of important functional changes in the hippocampus. Examples of ECM components is shown in Figure 13. Tenascin C is one example of a protein that is upregulated in our data set and that can be found in blood. Tenascin C is an extracellular matrix protein

implicated in the guidance of migrating neurons as well as axons during development, synaptic plasticity and neuronal regeneration. Serum levels of Tenascin C have been studied as a prognostic factor for survival after acute myocardial infarction (Sato A et al, 2012; Celik A et al, 2012).)

Interestingly, Tenascin C has been reported to be amongst a serum protein based profile with good diagnostic accuracy for AD (O'Bryant SE et al. 2010) and its level in serum was very recently found to significantly relate with activities of daily living in AD (Hall JR, 2012). Thus, our identification of increased levels of Tenascin C at early stages of AD brain pathology in the hippocampus supports our approach to relate early pathological changes in brain with early changes in bodily fluids, e.g., blood and/or serum for AD diagnosis.

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