WO2014065669A1 - Méthodes de traitement et de diagnostic d'un déclin cognitif et d'un trouble de la mémoire - Google Patents

Méthodes de traitement et de diagnostic d'un déclin cognitif et d'un trouble de la mémoire Download PDF

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WO2014065669A1
WO2014065669A1 PCT/NL2013/050756 NL2013050756W WO2014065669A1 WO 2014065669 A1 WO2014065669 A1 WO 2014065669A1 NL 2013050756 W NL2013050756 W NL 2013050756W WO 2014065669 A1 WO2014065669 A1 WO 2014065669A1
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ecm
hippocampus
proteins
individual
protein
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PCT/NL2013/050756
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August Benjamin Smit
Ronald Ernst VAN KESTEREN
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Stichting Vu-Vumc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • 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.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • A6 6- amyloid
  • APP ⁇ -amyloid precursor protein
  • neurofibrillary tangles which are intraneuronal aggregates of
  • hyperphosphorylated tau protein (Braak, de Vos et al. 1998; Selkoe 2001).
  • 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).
  • early memory impairments in human AD patients are not temporally correlated with the formation of ⁇ 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.
  • 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.
  • ECM extracellular matrix
  • 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.
  • ECM extracellular matrix
  • said one or more compounds treat at least one cognitive or memory related symptom of said dementia.
  • the alteration of the amount of or the composition of the ECM in the hippocampus increases synaptic plasticity in the hippocampus.
  • 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.
  • 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.
  • ECM extracellular matrix
  • 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.
  • ECM extracellular matrix
  • said rodent is a mouse model of Alzheimer's disease.
  • said one or more compounds alter the amount of or the composition of an ECM protein or hyaluronic acid.
  • the ECM protein is selected from a chondroitin sulfate
  • proteoglycans more preferably aggrecan, brevican, neurocan, NG2, versican and phosphacan, more preferably from versican.
  • said one or more compounds in not a metalloprotease or a metalloprotease inhibitor.
  • said one or more compounds is not MMP-9, MMP-2, MT1-MMP or an inhibitor of TIMP-1.
  • said compound is not a chondroitinase.
  • said compound is not a CD44 inhibitor.
  • the impairment in memory or cognition is due to excessive ECM.
  • the dementia is Alzheimer's disease (AD).
  • AD is in the early stages of AD, i.e., significant ⁇ plaques have not yet accumulated in the hippocampus of said individual.
  • 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.
  • the ECM preferably an ECM protein or hyaluronic acid as described herein
  • 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 ECM
  • ECM component is not Tenasin C.
  • a method of classifying an individual as having or being at risk of developing age-dependent cognitive decline and/or memory impairment, or dementia comprising determining the level of two or more extracellular matrix (ECM) components in the ECM
  • components as compared to a reference indicates that said individual has or is at risk of developing a cognitive, memory, or dementia.
  • the ECM component whose level is determined is an ECM protein, preferably selected from Versican and CD44.
  • 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.
  • 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.
  • 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.
  • said method comprises performing an immunoassay to determine the level of one or more ECM components in the hippocampus.
  • FIG. 1 Schematic representation of the 8-plex iTRAQ approach.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 7 Increased expression of ⁇ / ⁇ 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.
  • 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.
  • 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.
  • FIG. 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
  • FIG. 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
  • WFA-positive neurons were particularly high in the subiculum and the adjacent tip of the CAl region (DAPI: blue; WFA: green).
  • FIG 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.
  • ChABC ChABC
  • 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.
  • AD Alzheimer's disease
  • synaptophysin in the hippocampus and the association cortices (Dickson, Crystal et al. 1995; Sze, Troncoso et al. 1997; Reddy, Mani et al. 2005).
  • 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).
  • ECM Developmental and activity-dependent plasticity of the ECM are known to be important in the regulation of synaptic function.
  • 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.
  • 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.
  • PNN perineuronal nets
  • 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.
  • chondroitin sulfate proteoglycans e.g., aggrecan, brevican, neurocan, NG2, versican and phosphacan
  • tenascins e.g., Tnc and Tnr
  • hyaluronan link proteins e.g., Haplnl
  • 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,
  • HA hyaluronic acid
  • 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 are a family of enzymes which degrade hyaluronic acid.
  • MMP tissue inhibitor of metalloproteinase family
  • TIMPl tissue inhibitor of metalloproteinase family
  • 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 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
  • 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. ⁇ -aminopropionitrile (BAPN) is an enzyme that cross-links collagens or elastins in the ECM.
  • 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.
  • 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.
  • ECM extracellular matrix
  • 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.
  • 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).
  • the dementia is Alzheimer's Disease.
  • AD Alzheimer's Disease
  • MMSE Mini Mental State Examination
  • PET scans are also used in conjunction with mental status testing.
  • 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.
  • a composition as described herein is administered to an individual before amyloid plaques have developed in the hippocampus.
  • the alteration of the amount of or the composition of the ECM in the hippocampus increases synaptic plasticity in the hippocampus.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 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
  • 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
  • said compounds decrease the amount or composition of one or more chondroitin sulphate proteoglycans.
  • 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.
  • 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;
  • chondroitin sulphate proteoglycans preferably selected from aggrecan, brevican, neurocan, NG2, versi
  • 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.
  • chondroitin as e ABC, hyaluronidase, a matrix metalloprotease, neurotrypsin, Tissue-type plaminogen activator (tPA), an antibody that bind sulphation motifs of CSPGs.
  • tPA Tissue-type plaminogen activator
  • the compound decreases the function or expression of one or more chondroitin sulphate proteoglycans (CSPGs).
  • CSPGs chondroitin sulphate proteoglycans
  • 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
  • XT-1 Xylosytransferase-1
  • chondroitin 4 Xylosytransferase-1 (XT-1), and an inhibitor of chondroitin 4
  • said ChABC is a mammalian ChABC, more preferably a human ChABC.
  • the ChABC can be any enzyme having
  • the compound promotes the degradation or destabilzation of the ECM.
  • 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.
  • the compound decreases the production of the ECM, in particular decreases the production of ECM proteins and/or decreases post-translational modification thereof.
  • 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.
  • CSPGs chondroitin sulphate proteoglycans
  • 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
  • the compound is ChABC or an inhibitor of V-can or Tenascin-C.
  • the compound is ChABC or an inhibitor of V-can.
  • the compound is not Tenascin-C and/or a lysyl oxidase inhibitor.
  • the compound is not ChABC or a CD44 inhibitor.
  • the compound is not Tenascin-C, a lysyl oxidase inhibitor, ChABC, or a CD44 inhibitor.
  • the compound is not Tenascin-C, a lysyl oxidase inhibitor, or ChABC.
  • Inhibitors include polypeptides, small molecules (e.g., ⁇ -am nopropionitrile), and nucleic acid based inhibitors.
  • the inhibitor of a protein is a nucleic acid molecule such as an antisense oligonucleotide, an RNA
  • the compounds are nucleic acids encoding polypeptides in which the polypeptide is membrane anchored.
  • 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.
  • tPA Tissue-type plaminogen activator
  • 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.
  • said nucleic acids also comprise a hippocampal specific promoter.
  • 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.
  • ECM extracellular matrix
  • the protein and the membrane anchor are from different proteins, i.e., a chimeric protein is expressed.
  • the compound contains an endogenous membrane anchor (e.g., GPI-linked MMP17, type I transmembrane MMP14, and type II transmembrane MMP23).
  • 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.
  • the polypeptide comprises a GPI-signal peptide.
  • 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 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.
  • 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.
  • 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
  • the compound is an inhibitor of a protein.
  • 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.
  • the nucleic acid molecule is an antisense oligonucleotide.
  • Antisense oligonucleotides 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.
  • the nucleic acid molecule is an RNAi molecule, i.e., RNA
  • 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.
  • shRNA small hairpin RNA
  • stem loop is a type of siRNA.
  • these shRNAs are composed of a short, e.g.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • siRNA molecules 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).
  • 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.
  • stem- loop structure(s) derived from a specific endogenous miRNA
  • 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.
  • 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.
  • a gene delivery 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)
  • 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,
  • 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.
  • 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.
  • 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.
  • the inhibitors described herein are antibodies, e.g., antibodies that block the sulfation motifs of CSPGs.
  • 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.
  • 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.
  • a pharmaceutically acceptable carrier filler, preservative, adjuvant, solubilizer, diluent and/or excipient is also provided.
  • Suitable excipients comprise polyethylenimine (PEI) or similar cationic polymers, including polypropyleneimine or
  • PECs polyethylenimine copolymers
  • ExGen 500 synthetic amphiphils
  • SAINT- 18 synthetic amphiphils
  • lipofectinTM lipofectinTM
  • DOTAP DOTAP
  • viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, to a cell.
  • a compound is provided directly to the
  • 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)
  • an incision may be made in the scalp to expose the patient's 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.
  • composition required.
  • 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
  • compositions may be any composition 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.
  • ECM extracellular matrix
  • a preferred composition for the treatment of cognitive decline or memory impairment comprises a cholinesterase inhibitor (ChEIs).
  • ChEIs cholinesterase inhibitor
  • 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.
  • 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.
  • said individual is in Braak stages 1-2, more preferably in stage 1.
  • 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.
  • ECM extracellular matrix
  • 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.
  • said ECM component is not Tenasin C.
  • the method comprises determining the level of two or more extracellular matrix (ECM) components in the hippocampus of said individual.
  • said method comprises determining the level of Tenasin C and Versican or Tenasin C and CD44.
  • 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.
  • 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.
  • the ECM-binding compound is a small molecule, a peptide, a protein, or an antibody.
  • 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.
  • 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.
  • the ECM-binding compound is Wisteria floribunda agglutin (WFA), which labels chondroitin sulfates.
  • 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 ECM
  • said ECM-binding compound further comprises a label allowing for its detection.
  • 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.
  • Radio-tracer 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
  • 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.
  • 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.
  • 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.
  • 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.
  • assays using other ECM component binders may also be used.
  • 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).
  • SPR surface plasmon resonance
  • the method comprises the detection of Versican or a Versican peptide with a Versican specific antibody.
  • the method comprises the detection of CD44 or a CD44 peptide with a CD44 specific antibody.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • an element means one element or more than one element.
  • 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.
  • treating includes prophylactic and/or therapeutic treatments.
  • 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).
  • 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;
  • 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.
  • 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.
  • the lyophilized iTRAQ labeled samples were separated in the first dimension by strong cation exchange column (2.1x150 mm polysulfoethyl A column,
  • FDR false discovery rate
  • 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.
  • the One-Class Time Course Analysis option implemented in the Significance Analysis of Microarrays (SAM) package was used to determine significant regulation over time.
  • 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.
  • 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).
  • SD standard deviations
  • 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;
  • 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.
  • Proteomic analysis identifies 25 proteins that are significantly regulated over time
  • 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).
  • cortical cytoskeleton e.g., Acnt4, Nefl, Nefm
  • non-membrane bound organelles e.g., Kif5c, Rps4x
  • microtubule-based processes and movement e.g., Tubb4, Tubb5, Kif5c
  • SD standard deviations
  • nucleocytoplasmic transport establishment of protein localization, synaptic vesicle transport, cell junction, extracellular structure organization, and ATPase activity.
  • High variability proteins 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
  • neurodegeneration-related alpha-synuclein (Snca) protein was found to be highly variable.
  • 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 ⁇ 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).
  • 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 ⁇ / ⁇ and ApoE in APP-PSl mice
  • 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).
  • a new group of three months-old APP-PSl animals and WT littermates 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.
  • Adeno-associated virus 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.
  • mmu00980 Metabolism of xenobiotics by cytochrome P450 UP mmu00982:Drug metabolism UP
  • G-protein 4 Gnaq, Gnall, Gnaol, Gnaz 0.343
  • 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
  • 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).
  • 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.
  • Citron M., T. Oltersdorf, et al. (1992). "Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.” Nature 360(6405): 672-4.

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Abstract

La présente invention concerne des traitements d'un déclin cognitif et/ou d'un trouble de la mémoire fondés sur la réduction de la matrice extracellulaire dans l'hippocampe. L'invention concerne des compositions améliorées pour de tels traitements. L'invention porte également sur des méthodes de détection permettant le diagnostic précoce d'un déclin cognitif et/ou d'un trouble de la mémoire, fondées sur une augmentation de la matrice extracellulaire dans l'hippocampe. Les méthodes de traitement et de diagnostic sont particulièrement utiles dans le domaine de maladie d'Alzheimer.
PCT/NL2013/050756 2012-10-26 2013-10-28 Méthodes de traitement et de diagnostic d'un déclin cognitif et d'un trouble de la mémoire WO2014065669A1 (fr)

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US20200046277A1 (en) * 2017-02-14 2020-02-13 Yuen Lee Viola Lam Interactive and adaptive learning and neurocognitive disorder diagnosis systems using face tracking and emotion detection with associated methods
CN113462769A (zh) * 2021-08-05 2021-10-01 中国医学科学院医药生物技术研究所 抑制剂/CaMKII体系及作为生物标志物中的应用

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