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WO2008027017A1 - Modulation de l'activité et/ou affection neurales - Google Patents

Modulation de l'activité et/ou affection neurales Download PDF

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Publication number
WO2008027017A1
WO2008027017A1 PCT/SG2007/000283 SG2007000283W WO2008027017A1 WO 2008027017 A1 WO2008027017 A1 WO 2008027017A1 SG 2007000283 W SG2007000283 W SG 2007000283W WO 2008027017 A1 WO2008027017 A1 WO 2008027017A1
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Prior art keywords
app
protein
tag
fragment
derivative
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Zhi Cheng Xiao
Quan-Hong Ma
Shao Li
Gavin Stewart Dawe
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Singapore Health Services Pte Ltd
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Singapore Health Services Pte Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1716—Amyloid plaque core protein
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention generally relates to the field of medical and biological science.
  • the present invention relates to methods to modulate neural conditions, activities and/or events.
  • Amyloid precursor protein is well known as a molecule that is involved in the pathogenesis of Alzheimer's disease (AD) and of other forms of dementia, such as Down's syndrome.
  • AD Alzheimer's disease
  • a ⁇ beta amyloid
  • a ⁇ A ⁇ Deposition of A ⁇ , has been suggested to be related to the pathology of AD and associated with other pathological markers of AD conditions such as cell death in the central nervous system (CNS), accumulation of amyloid plaques, and the appearance of neurofibrillary tangles. Reduction or prevention of A ⁇ deposition is widely believed to be desirable for slowing the progress of, or preventing the development of, AD. While reduction of A ⁇ deposition is thought to be desirable, there is no teaching or suggestion in the literature on how this may be achieved.
  • the entorhinal cortex from where the perforant pathway originates, is one of the most severely affected areas, particularly at the earliest stages of the disease.
  • electrical activity within the pathway modulates interstitial fluid ⁇ -amyloid (A ⁇ ) levels, which can be blocked by tetrodotoxin (TTX), a specific sodium channel blocker (Cirrito et al, 2005).
  • TTX tetrodotoxin
  • any contribution to the understanding of the role of APP in the brain will be useful and will add to knowledge of neural functions and/or events, as well as to treatment of neurological conditions.
  • the present invention addresses the problems above, and in particular, provides new methods to modulate at least one biological or medical condition, activity (function) and/or events.
  • the present invention provides a method of treating at least one medical condition in a subject, the method comprising modulating the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment.
  • the modulating may be by decreasing the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment.
  • the modulation of APP, its derivative or a fragment thereof may comprise modulating at least one molecule, protein, and/or peptide that interacts with amyloid precursor protein (APP), APP derivative or APP fragment.
  • the modulation may comprise modulating at least one cellular component that interacts with amyloid precursor protein (APP), APP derivative or APP fragment.
  • the at least one cellular component may be selected from TAG-1 , sodium channels, and sodium channel complex protein.
  • the modulation may comprise modulating at least one cellular component selected from Caspr2, NgR, TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ NG2 and Go protein(s).
  • the modulating according to the invention comprises administering at least one pharmaceutically and/or therapeutically effective amount of amyloid precursor protein (APP), APP derivative or APP fragment.
  • APP amyloid precursor protein
  • APP derivative or APP fragment is A ⁇ , soluble APP (sAPP), APP-695, AICD or VTPEER peptide.
  • the modulating may comprise administering a pharmaceutically and/or therapeutically effective amount of at least one drug, protein, peptide, antibody or molecule that interacts with amyloid precursor protein (APP), APP derivative or APP fragment, TAG-1 , sodium channels, or sodium channel complex protein(s).
  • the modulating may also comprise administering a pharmaceutically and/or therapeutically effective amount of at least one drug, protein, peptide, antibody or molecule that interacts with amyloid precursor protein (APP), APP derivative or APP fragment, TAG-1 , sodium channels, TAG-1 complex protein(s), sodium channels complex protein(s), Caspr2, NgR, potassium channels, TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ and NG2.
  • the modulating may also comprise administering a pharmaceutically and/or therapeutically effective amount of at least one compound selected from the group consisting of TAG-1 , anesthetic(s), toxin(s) and Go protein(s).
  • the modulating may comprise administering a pharmaceutically and/or therapeutically effective amount of at least one drug or molecule selected from local anaesthetic(s), veratridine, batrachotoxin, scorpion toxins, sea anemone toxin, conus toxins, saxitoxin, tetrodotoxin, antiepileptic drug(s), antiarrhythmic drug(s), antibodies to sodium channel proteins, antibodies to Caspr, and peptide fragments of APP.
  • a drug or molecule selected from local anaesthetic(s), veratridine, batrachotoxin, scorpion toxins, sea anemone toxin, conus toxins, saxitoxin, tetrodotoxin, antiepileptic drug(s), antiarrhythmic drug(s), antibodies to sodium channel proteins, antibodies to Caspr, and peptide fragments of APP.
  • the modulation of amyloid precursor protein (APP), APP derivative or APP fragment may be to prevent and/or reduce beta amyloid (A ⁇ ) deposition, improve and/or facilitate nervous tissue repair, improve and/or facilitate myelination, to modulate stem cell function(s) and/or to modulate sodium channel function(s).
  • the at least one medical condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis, brain injury, spinal cord injury, axonal injury-related disorders, epilepsy, neurodegenerative disorders and demyelination disorders.
  • the modulating may result in modulation of axonal conduction of action potential(s) for the treatment of pain and/or epilepsy.
  • the modulating may result in the induction of analgesia and/or local anaesthesia.
  • the APP derivative may be soluble APP (sAPP) and the APP fragment may be beta amyloid (A ⁇ ).
  • sAPP soluble APP
  • a ⁇ beta amyloid
  • the present invention also provides a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, a neurological condition in a subject, the method comprising: (a) providing at least one sample from a subject; (b) determining the expression or activity of amyloid precursor protein (APP), APP derivative or APP fragment; (c) comparing the expression or activity of amyloid precursor protein (APP), APP derivative or APP fragment, with that of at least one control, a difference in expression and/or activity indicating the presence of, or predisposition to, and/or severity of a neurological condition in the subject.
  • a method of monitoring the efficacy of a treatment for a neurological condition in a subject comprising: (a) providing at least two samples from a subject, each sample obtained at different time points; (b) determining the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment; and (c) comparing the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment, in the at least two samples.
  • a method of prognosticating the outcome of a neurological condition in a subject comprising: (a) providing at least one sample from a subject; (b) determining the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment; (c) comparing the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment, with that of at least one control, a difference in expression and/or activity (function) indicating the prognosis of a neurological condition in the subject.
  • a method of inducing analgesia and/or anesthesia in a subject comprising modulating the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment.
  • the modulating may comprise decreasing the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment.
  • the present invention also provides an in vitro or in vivo method of modulating proliferation, differentiation and/or function (activity) of at least one stem cell, the method comprising modulating the expression and/or activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment, in the at least one stem cell.
  • APP amyloid precursor protein
  • the present invention also provides a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, at least one condition related to beta amyloid (A ⁇ ) deposition and/or sodium channel dysfunction in a subject, the method comprising: (a) measuring the nerve and/or axon conduction velocity in the subject; (b) comparing the nerve and/or axon conduction velocity with that of at least one control, a difference in velocity indicating the presence of, or predisposition to, and/or severity of at least one condition related to A ⁇ deposition and/or sodium channel dysfunction in the subject.
  • the method may be for detecting and/or quantitating the presence of, predisposition to, and/or severity of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • the present invention also provides an in vitro or in vivo method of modulating at least one activity (function) of amyloid precursor protein (APP), APP derivative or APP fragment, comprising administering at least one drug, protein, peptide, antibody or molecule that interacts with amyloid precursor protein
  • APP amyloid precursor protein
  • APP derivative or APP fragment comprising administering at least one drug, protein, peptide, antibody or molecule that interacts with amyloid precursor protein
  • the modulating may comprise administering a pharmaceutically and/or therapeutically effective amount of at least one compound selected from the group consisting of TAG-1 , anesthetic(s), toxin(s) and Go protein(s).
  • the method may be for treating at least one condition selected from the group consisting of Alzheimer's disease, multiple sclerosis, brain injury, spinal cord injury, axonal injury-related disorders, epilepsy, neurodegenerative disorders and demyelination disorders.
  • the present invention also provides an in vitro or in vivo method of modulating sodium channel(s) activity(ies) (function/s) comprising administering at least one drug, protein, peptide, antibody or molecule that interacts with amyloid precursor protein (APP), APP derivative or APP fragment, TAG-1, sodium channel(s), or sodium channel complex protein(s).
  • the method may be for modulating at lease one APP activity (function), and/or for the treatment of at least one neurological disorder.
  • the method may be for the treatment of acute and/or chronic pain disorder(s), induction of analgesia, treatment of epilepsy and/or convulsion(s), treatment of disorder(s) of the rhythm of the heart, and/or induction of anaesthesia and/or analgesia.
  • the present invention also provides at least one pharmaceutical composition for treating at least one APP and/or sodium channel related medical condition comprising at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1 , , a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s), toxin(s) and Go protein(s); and optionally at least one pharmaceutically acceptable diluent, carrier and/or excipient.
  • the medical condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis, brain injury, spinal cord injury, axonal injury-related disorders, epilepsy, neurodegenerative disorders and demyelination disorders.
  • the present invention also provides use of at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to
  • APP TAG-1 , at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s), toxin(s) and Go protein(s), for the preparation of a medicament for the treatment of an APP and/or sodium channel related medical condition.
  • the present invention also provides a compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1 , at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s), toxin(s) and Go protein(s) for the treatment of an APP and/or sodium channel related medical condition
  • the present invention also provides a kit comprising at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1 , at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s) and toxin(s), for the detection and/or
  • the APP derivative or fragment is A ⁇ , soluble APP (sAPP), App-695, AICD or VTPEER peptide.
  • Figure 1 shows that APP is expressed by axons during myelination. It also shows the interaction between APP and TAG-1 , Caspr2, NgR, and K+ channels.
  • Figure 2 shows that APP is expressed at nodes of Ranvier. Interaction with Sodium channels places APP in the context of TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, Omgp, RPTO ⁇ and NG2 complexes.
  • FIG. 3 shows that TAG-1 is expressed in the stem cell niche and that APP interacts with TAG-1.
  • Figures 4-8 show that APP locates at NORs in the CNS.
  • Figure 4 shows that APP was detected at NORs in the spinal cord
  • a-k Normal adult rat spinal cord were prepared for double IF, using polyclonal antibodies to APP (green; a-k) and monoclonal antibodies against Kv1.2 (Kch; red; a d, f, and g) or Tn-R (red; b, h j, and k) or Na+ channels (Nach; red; c), respectively
  • g and k Orthogonal slices. Scale bars: 10 ⁇ m in a-c, f, g, j, and k.
  • Figures 5(a, b) show that APP evenly distributed along sciatic nerve.
  • SN Normal adult rat sciatic nerves
  • polyclonal antibodies to APP Green
  • monoclonal antibodies against NF200 red; a
  • Kv1.2 red; b
  • Figures 5(c, d, e) show disappearance of APP clustering in the spinal cord of APP knock-out mice.
  • Adult spinal cord from APP knock-out mice (KO) was prepared for double IF, using polyclonal antibodies to APP (Green; a) or Caspr (Green; b and c) and monoclonal antibodies against Tn-R (red; a and b) and Nach (red; c), respectively.
  • Scale bars 10 ⁇ m in a, b.
  • Figure 6 shows the results of immunohistochemical studies of longitudinal sections of rat spinal cords at P6, P10, P15, and P21. Double labellings using antibodies against pan-Na+ channels (Nach, red; a) and K+ channel (Kch, red; b to d) showed that APP clustering commences at P10 and becomes prevalent by P15. Insert boxes show images with higher magnification from the respective lower magnification images.
  • Figure 7 shows quantification of double IFs of APP and K+ channels performed in spinal cords.
  • Scale bars 10 ⁇ m in a-d.
  • Figures 9-12 show myelination in APP knock-out and transgenic mice (Scale bars: 0.5 ⁇ m in Aa, Ab, Ca, and Cb; 5 ⁇ m in Ba, Bb. Error bars represent +SEM in Ac, Bc, and Cc. ** p ⁇ 0.001).
  • Figure 9 shows the results of EM studies in cross sections revealed the ultrastructure of myelin sheaths in spinal cords (SC) of wild-type littermates (WT; a) versus APP knock-out mice (KO; b) (all aged 3 months). The myelin thickness as indicated by g ratio (c) was significantly reduced in APP knock-out mice, in comparison with wild-type littermates.
  • FIG 10 shows the results of EM studies on the ultrastructure of myelin sheaths in sciatic nerve (SN).
  • Cross sections revealed that wild-type littermates (WT; a) versus APP knock-out mice (KO; b) (all aged 3 months).
  • the myelin thickness as indicated by g ratio (c) was significantly reduced in APP knock-out mice, in comparison with wild-type littermates.
  • Figure 11 shows the results of EM studies in cross sections revealed the ultrastructure of myelin sheaths in spinal cords (SC) of wild-type littermates (WT; a) versus APP transgenic mice (TG; b) (all aged 3 months).
  • the myelin thickness as indicated by g ratio (c) was significantly increased in APP transgenic mice, in comparison with wild-type littermates.
  • Figure 12 shows the results of APP knock-out and overexpression showed the opposite effects on both axon (a) and fiber (b) diameters and the myelin thickness as indicated by g ratio (c), in mice after normalizing with wild-type littermates, respectively.
  • Figures 13-16 show that APP modulates the expression of Na+ channel Nav1.6 in Xenopus oocytes.
  • Figure 13 illustrates the effects of APP on voltage-gated sodium channels. a.
  • a shows representative examples of Na+ current responses elicited by depolarizing voltage steps.
  • b shows normalized current-voltage relationships of Na+ currents in oocytes expressing Na+ channels alone (open circles) and together with APP (closed circles).
  • c shows activation of Na+ currents in oocytes expressing Na+ channels alone (open circles) and together with APP (closed circles).
  • d shows normalized inactivation of Na+ currents in oocytes expressing Na+ channels alone (open circles) and together with APP (closed circles).
  • e shows recovery from inactivation measured using a two-pulse protocol with a variable interval in oocytes expression sodium channels alone (open circles and together with APP (solid circles).
  • Figures 17-18 show electrophysiological properties in APP-knock-out mice.
  • Figure 17 shows whole cell recordings of voltage-gated Na+ currents in acutely dissociated DRG neurons, a, Na+ current responses elicited by depolarizing voltage steps in a neuron from an APP knockout mouse (top). Normalized current-voltage curves of peak Na+ currents in wild-type mice (solid circles) and
  • APP knockout mice (open circles).
  • pF picofarads
  • b activation (circle symbols) and steady-state inactivation (triangle symbols) curves of Na+ currents in neurons from wild-type mice (dash line) and APP knockout mice
  • Figure 18 shows the determination of conduction velocities of compound action potentials in APP-knock-out and wild-type mice.
  • Significance values were calculated using the Student's t test and were as follows: 24°C, p ⁇ 0.05; 32°C, p ⁇ 0.05; and 37°C, p ⁇ 0.005. Error bars represent +SEM.
  • Figure 19A shows APP expression during development. Protein extracts of rat whole brains at various postnatal developmental stages (PO, P5, P7, P8, P10, P15, P21 , P30 and adult) were subjected to Western blotting analyses using antibodies against APP and ⁇ -tubulin (as loading control).
  • Figure 19B shows that APP clustering was disrupted in the spinal cord of PLP transgenic mice (arrow). APP is still clustering at node of Ranvier in the unaffected myelinated fiber (arrowhead).
  • Adult spinal cords from both mice were prepared for double IF, using polyclonal antibodies to APP (green) and monoclonal antibodies against K+ channels (red) respectively. Scale bars: 10 ⁇ m.
  • Figures 20-22 show effects on axon and fiber diameters. (Note that increment of g ratio in KO mice is significant in almost all axons. ** p ⁇ 0.001 ; *p ⁇ 0.05. Error bars represent +SEM.)
  • Figure 20 shows that both axon (a) and fiber (b) diameters were significantly reduced in APP knock-out mice, in comparison with wild-type littermates.
  • the distribution of the g ratio, representing the myelin thickness, in the spinal cord (c) was plotted against different axon diameters, comparing APP KO to WT mice.
  • Figure 21 shows the distribution of G ratio representing the myelin thickness of sciatic nerve was plotted against different axon diameters, comparing in APP KO and WT mice.
  • Figure 22 shows that both axon (a) and fiber (b) diameters were significantly increased in APP transgenic mice, in comparison with wild-type littermates.
  • Figure 23 shows effect of co-expression of APP on peak Na+ currents of oocytes expressing the Nav1.6 with a, ⁇ subunit; b, ⁇ + ⁇ 1 subunit; c, ⁇ + ⁇ 2 subunit; and d, ⁇ + ⁇ 1 + ⁇ 2 subunit; ** p ⁇ 0.005.
  • Figure 24 shows a western blot analyses of protein extracts from the brains of adult APP +/+ , APP*' ' , and APR A mice. Antibodies used were directed against Nav1.6 and ⁇ -tubulin (as loading control).
  • Figure 25 shows a schematic representation of APP (Full-length APP695; SEQ ID NO: 2, cDNA; SEQ ID NO: 3, peptide), APP intracellular domain (AICD50; SEQ ID NO: 4, cDNA; SEQ ID NO: 7, peptide), APP C31 domain (C31 ; SEQ ID NO: 8, cDNA; SEQ ID NO: 10, peptide), APP Go protein binding domain (GOBD; SEQ ID NO: 11, cDNA; SEQ ID NO: 14, peptide), Fe65 binding domain (Fe65BD; SEQ ID NO: 15, cDNA; SEQ ID NO: 18, peptide) and VTPEER peptides (Val 667 -Arg 672 ; SEQ ID NO: 19, cDNA; SEQ ID NO: 22, peptide).
  • Figure 26 shows the effects of distinct APP intracellular domains and peptides on voltage-gated sodium channels.
  • Figure 27 shows the effects of Go protein on the APP-induced sodium current expression.
  • a Sodium channels and APP alone (open squares) and together with a G203T dominant negative mutant of the Go protein ⁇ subunit (GoDN; closed circles).
  • b Sodium channels and AICD alone (open squares) and together with GoDN (closed circles).
  • c Sodium channels and the Go protein-binding domain (GB) of APP alone (open squares) and together with GoDN (closed circles).
  • GB Go protein-binding domain
  • GB Go protein-binding domain
  • Fe65BD Fe65 binding domain
  • APP alone open squares
  • GoDN closed circles
  • e Sodium channels and the Fe65 binding domain (Fe65BD) of APP alone (open squares) and together with GoDN (closed circles).
  • e Sodium channels and the C31 domain (C31) of APP alone (open squares) and together with GoDN (closed circles).
  • f Sodium channels and the VTPEER
  • Figure 29 shows the relationship between the amplitude of normalized sodium currents and the APP concentration.
  • Nav1.6 was co-expressed with full length APP in various molar ratios in oocytes.
  • the graph shows the normalized mean peak sodium current amplitude
  • Figure 30 illustrates that APP and TAG-1 are binding partners.
  • TAG-1 transfected CHO cells (a), but not F3 transfected CHO cells (d) and CHO cells (e), adhered to APP-Fc protein spots. Adhesion of TAG-1 transfected CHO cells to APP-Fc was blocked by antibodies against TAG-1 (b) and APP (c).
  • APP transfected CHO cells (f, g, h, j and I) 1 but not CHO cells (i, k, m, and n), adhered to spots coated with TAG-1-Fc protein (f), TAXIg-GST G), and TAXFNII-GST (I), but not GST (n). Adhesion of APP transfected CHO cells to TAG-1 -Fc was blocked by antibodies against TAG-1 (g) and APP (h). Bar in n is 20 ⁇ m (for a-n). ** p ⁇ 0.001.
  • Figure 31 shows that (a) APP and TAG-1 associate as a protein complex. Co- immunoprecipitation of APP with TAG-1. Mouse brain lysates were co- immunoprecipitated using anti-TAG-1 antibodies and non-immune IgG and probed with anti-APP antibody. Reciprocal assays used anti-APP antibody or APP-Fc to capture the protein complex and anti-TAG-1 antibodies to detect the binding partner, (b) GST Pulldown assay to analyze the interaction between TAX and APP. Mouse brain, APP/CH0, and CHO cell lysates were precipitated using TAXFNIII-GST, TAXIg-GST 5 and GST and probed with anti-APP antibody.
  • Figure 32 shows that AICD transcriptional activity in TAG-1- and TAX- transfected CHO cells and expression of APP and TAG-1 in the fetal neural stem cell niche.
  • A (a) CHO cells and L1 (CHOL1), TAX (CHOTAX) and TAG-1 (CHOTAG 1) transfected CHO cells were transiently co-transfected in 24-well culture dishes with pG5E1 B plasmid, APP-Gal4-responsive luciferase reporter gene, Fe65 plasmid, and luciferase internal control plasmid.
  • Figure 33 shows double immunostaining for nestin (red, a, a', d, and d'; c, c ! , f, and f are the merged images) and APP (green, b, b', c, and c') or TAG-1 (green, e, e', f, and f ) in the walls of the lateral ventricles in E14 mouse brain.
  • Double immunostaining for APP green, h and h'; i and i' are the merged images
  • TAG-1 red, g, g', i, and i'
  • Bars in c, f, and i are 50 ⁇ m (for a, b, d, e, g, and h); Bars in c', f , i' are 20 ⁇ m (for a', b', d ⁇ e ⁇ g ⁇ and h').
  • Figure 34 shows the expression of APP and TAG-1 in neural progenitor cells (NPCs).
  • NPCs isolated from the lateral ventricles of E14 mouse brain were double-stained for APP (green; a' and red; c' and e) and/or TAG-1 (green; b' and e' and red; d') and with neural precursor cell markers, nestin (red; a and b) or Sox2 (green; c and d).
  • a" to e" are merged images.
  • Mouse brain samples and the NPCs lysate were blotted with antibodies against TAG-1 and APP, respectively (f).
  • Bars are 20 ⁇ m in a" (for a and a'); b" (for b and b'); c" (for c and c'); d" (for d and d'); and e" (for e and e').
  • Figure 35 shows the neurogenesis in APP and TAG-1 deficient mice.
  • NPCs were isolated from APP +/+ mice (APP WT) and APP -/- mice (APP KO). After 7-8 days in vitro culture differentiation, the cells were double-stained for TUJ 1 (a and b) or MAP2 (d) and DAPI (a and b). The number of TUJ 1 (c) and MAP2 (d) positive cells were counted and expressed as a percentage of the total number of DAPI positive cells. Bar in b is 100 ⁇ m (for a).
  • NPCs isolated from TAG-1 +/+ mice (TAG-1 WT) and TAG-1 -/- mice (TAG-1 KO) were double-stained for TUJI (a and b) or MAP2 (d) and DAPI (a and b).
  • the number of TUJ 1 (c) and MAP2 (d) positive cells were counted and expressed as a percentage of the total number of DAPI positive cells.
  • Bar in b is 100 ⁇ m (for a). ** p ⁇ 0.001. Error bars represent standard deviations.
  • Figure 36 shows TAG-1 triggered AICD transcriptional activity in NPCs, neurogenesis in TAG-1 /APP double deficient mice and a putative model of the TAG-1 /APP pathway.
  • NPCs isolated from APP-/-/TAG-1-/-mice were transiently co-transfected with pG5E1B plasmid, a APP-Gal4-responsive luciferase reporter gene, Fe65 plasmid, and luciferase internal control plasmid (a).
  • the transfected NPCs were cultured in 24-well culture dishes substrate-coated with laminin, F3-Fc, LI-Fc, and TAGI-Fc as indicated. Normalized luciferase activities in whole-cell lysates were determined in triplicate and expressed relative to the activity in lysates prepared from laminin treated cells.
  • AICD transcriptional activity in APP-/-/TAG- 1-/- NPCs was significantly reduced by a ⁇ -secretase inhibitor (2 ⁇ M and 4 ⁇ M, respectively; b). ** p ⁇ 0.001. Error bars represent standard deviations.
  • NPCs isolated from AP PWTAG- 1+/+ mice (WT) (a) and TAG-1 -/-/APP-/- mice (b) were double-stained for TUJI and DAPI. The percent of TUJ1/DAPI positive cells were counted (e). ** p ⁇ 0.001. Error bars represent standard deviations. Bar in b is 100 ⁇ m (for a).
  • Figure 37 shows a putative model of the TAG-1/APP pathway. TAG-1 interacts with APP on the ceil surface to activate the APP signaling pathway through y- secretase-dependent cleavage. This is followed by the processing of APP and release of AICD into cytoplasm, where AICD may bind with Fe65 or other modifiers.
  • the complex further translocates to the nucleus, where it interacts with other transcription-related proteins, such as Tip60, and thereby activates target genes.
  • other transcription-related proteins such as Tip60
  • neurogenesis is negatively modulated by AICD transcriptional activity triggered by the TAG-1/APP interaction.
  • Medical or biological activity (function), event and/or condition - are events, activities (functions) and/or conditions in humans and/or animals. Since such events, activities (functions) and/or conditions are homologous or equivalent in both humans and animals, and/or share many similar causations, processes or pathways, the present invention may be applicable to either humans or animals or to both. Accordingly, events, activities (functions) and/or conditions referred to in humans also refer to homologous or equivalent events, functions or conditions in animals.
  • Neural/neurological activity (function) - Neural activities (functions) are activities (functions) controlled by the nervous system. These include the functioning of the nervous system, sensing of changes in both the internal and external environments, the interpretation of those changes and the coordination of responses. Neural activities (functions) may comprise of one or more neural events.
  • activity or “function” are assumed to have the same meaning and are used interchangeably. Further, any reference to the term in singular form, for example, “activity” or “function” also encompasses the plural term, that is, “activities” or “functions”.
  • Neural event - events taking place in the nervous system such as myelination, differentiation, growth, changes and cell death and includes generation and propagation of action potentials. Neural events can either be normal or abnormal.
  • Neurological condition the state of a part of, or the entire nervous system. It usually refers more to abnormal or disease conditions than normal conditions.
  • any reference to APP may also apply to any APP derivative as well as to any APP fragment which maintain the same, similar, equivalent or comparable biological activity (function) of APP.
  • APP activity (also indicated as "function” as defined above) relates to the APP biological activity (function).
  • function the neural/neurological activity (function), as defined above, of APP.
  • APP capacity to interact with other cellular proteins and/or exogen drug(s) or compound(s) and to modulate their or its activity (function).
  • APP capacity to modulate the activity (function) of sodium channel(s) and TAG-1.
  • the APP activity (function) also encompasses the cleavage of APP and the formation of the beta amyloid (A ⁇ ).
  • Modulating - varying or changing a state, level, system or event may be by increasing or decreasing the level or expression of a molecule in a system.
  • Expression - The conversion of a gene into a gene product which may be RNA or protein. Accordingly, the expression of a gene product may be controlled at two levels: at the transcription level or at the translation level, to obtain a change in the prevalence of the molecular product.
  • Cellular component Any part of a cell including structural and non-structural components.
  • a protein may also have other non-amino acid components such as carbohydrates or lipids associated with it or may be post-translationally modified (e.g. by glycosylation or phosphorylation).
  • a protein may be processed in or outside the cell (ie in vivo or in vitro) to obtain derivatives or cleaved to form one or more protein fragments.
  • a protein may be purified from natural sources or artificially synthesized.
  • a protein under the present invention includes proteins that are substantially identical to it. By “substantially identical", it is meant a protein exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% sequence identity to the reference protein.
  • a molecule is said to be homologous to a reference molecule if that molecule is able to exert or elicit a similar response in a cell or biological system as that exerted or elicited by the reference molecule.
  • a synthetic molecule having an active site similar to that of a biologically-active reference molecule may be able to exert or elicit a similar response in a cell or biological system without the synthetic molecule having to possess a similar overall chemical structure as that of the reference molecule.
  • Interacts/interaction - Two biological molecules are said to interact with one another when either of them causes a change in the other molecule or to itself.
  • the change may be a physical change or a change in biological properties. Accordingly, the interaction may be direct wherein the two molecules come into direct physical contact or the interaction may be indirect when the interaction is via one or more intermediate molecules.
  • Sodium channel complex refers to a complex comprising molecules which interact with sodium channel.
  • the molecules include but are not limited to Caspr2, NgR, potassium channels, Tn-R, Tn-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ , NG2 and Go protein(s).
  • TAG-1 complex refers to a complex comprising molecules which interact with TAG-1.
  • the molecules include but are not limited to APP, Caspr2, NgR, K+ channels, Tn-R, Tn-C, MAG, NogoA, phosphacan, F3, OMgp, RPTP ⁇ , NG2 and Fe65.
  • Myelinated axons in the central nervous system are characterized by their segmental structures: the node of Ranvier (NOR), paranode, juxtaparanode, and intemode.
  • NOR Ranvier
  • paranode paranode
  • juxtaparanode juxtaparanode
  • intemode axons in the central nervous system
  • PNS peripheral nervous system
  • TAG-1 This localization and interaction with TAG-1 reveals novel activities (functions) for APP. In particular, it reveals novel activities (functions) for APP in myelination.
  • the interaction with at least sodium channels and/or TAG-1 also provides novel target(s) for the modulation of APP function. It also provides novel targets for the modulation of A ⁇ deposition in Alzheimer's disease and other diseases associated to the modulation of APP, sodium channels or TAG-1. In particular, it provides novel target(s) for diseases related with A ⁇ deposition.
  • the inventors provide method(s) to modulate biological and/or medical events, activities (functions) and/or conditions, such as neural activities (functions), events and/or neurological conditions, by modulating the activity (function) and/or expression of APP, its derivative or a fragment thereof, or a molecule that interacts with APP, its derivative or a fragment thereof.
  • TAG-1 is an interacting partner of APP in the stem cell niche and accordingly provides novel target(s) for modulation of stem cell activity (function).
  • association of APP with TAG-1 and sodium channel(s) reveals numerous other targets for modulation of APP by the known association of TAG-1 and sodium channels with protein complexes.
  • protein complexes involve numerous molecules including Caspr2, NgR, K+ channels, TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ and NG2.
  • the findings also suggest novel means of modulating sodium channel activity (function) and hence axonal conduction that may have application in treatment of pain, in local anaesthesia, or in the treatment of neurological disorders such as epilepsy.
  • TAG-1 is localized on stem cells and in the stem cell niche in the brain.
  • novel targets for modulation of APP and its functions in health and disease e.g. to treat AD or to manipulate stem cells
  • novel activities (functions) of APP in myelination e.g. in the treatment of brain or spinal injury and MS.
  • the invention also provides novel target(s) for modulating sodium channel activity(ies) (function/s) (e.g. to achieve analgesia, local anaesthesia, or in the treatment of neurological disorders such as epilepsy).
  • APP is expressed by axons during myelination and interactions with TAG-1 places APP in the TAG-1 , Caspr2, NgR, K+ channel complex and reveals targets for modulation of myelination (e.g. in treatment of multiple sclerosis or spinal inury).
  • Figure 2 shows that APP is expressed at nodes of Ranvier and interactions with sodium channels places APP in the context of TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ and NG2 complexes and reveals both novel targets for modulation of APP activity (function) including amyloid deposition (e.g.
  • FIG. 3 shows that TAG-1 is expressed in the stem cell niche and that APP interacts with TAG-1 reveals novel targets for modulation of stem cell activity (function) (e.g. in cytotherapeutic brain repair).
  • the present inventors provide novel means to modulate at least (a) APP, its derivative or fragment thereof, (b) Myelination, (c) Sodium channel(s) and/or (d) stem cell(s).
  • the present invention provides the use of drugs, peptides or other molecules interacting with sodium channel(s) to modulate APP activity (function).
  • the modulation of APP function may be used to prevent amyloid deposition (e.g. treatment of AD), to improve and/or facilitate nervous tissue repair (e.g. treatment of spinal injury), to improve and/or facilitate myelination (e.g. in the treatment of MS), or to modulate stem cell activity (function) (e.g. for brain repair).
  • Examples of such drugs and molecules include but are not limited to local anaesthetics (e.g.
  • lidocaine veratridine
  • batrachotoxin scorpion toxins
  • sea anaemone toxin conus toxins
  • saxitoxin tetrodotoxin
  • antiepileptic drugs e.g. carbamazepine and phenytoin
  • antiarrhythmic drugs e.g. quinidine
  • antibodies to sodium channel proteins e.g. sodium channel proteins, and peptide sequences derived from APP.
  • the modulation of sodium channel activity may include modulation of APP expression and/or activity (function), treatment of acute or chronic pain disorders, induction of analgesia, treatment of epilepsy or convulsions, treatment of disorder of the rhythm of the heart, or induction of local anaesthesia.
  • drugs, peptides or other molecules interacting with TAG-1 may be used to modulate APP function.
  • the modulation of APP activity (function) may either be used to prevent amyloid deposition (e.g. treatment of AD), to improve and/or facilitate nervous tissue repair (e.g.
  • Such molecules include but are not limited to antibodies to TAG-1 and peptides derived from APP or Caspr.
  • TAG-1 or peptides which mimic part of the sequence of TAG-1 to modulate APP activity (function).
  • the modulation of APP activity (function) may either be used to prevent amyloid deposition (e.g. treatment of AD), to facilitate nervous tissue repair (e.g. treatment of spinal injury), to improve and/or facilitate myelination (e.g. in the treatment of MS), to modulate stem cell function (e.g. for brain repair), or to modulate sodium channel activity (function) (e.g. treatment of epilepsy or induction of local anaesthesia).
  • AD amyloid deposition
  • nervous tissue repair e.g. treatment of spinal injury
  • myelination e.g. in the treatment of MS
  • stem cell function e.g. for brain repair
  • sodium channel activity e.g. treatment of epilepsy or induction of local anaesthesia
  • Examples of such molecules include but are not limited to full length TAG-1 and peptides comprising subset of the TAG-1 protein sequence.
  • APP activity may either be used to prevent amyloid deposition (e.g. treatment of AD), to improve and/or facilitate nervous tissue repair (e.g. treatment of spinal injury), to improve and/or facilitate myelination (e.g. in the treatment of MS), to modulate stem cell function (e.g. for brain repair), or to modulate sodium channel activity (function) (e.g. treatment of epilepsy and/or induction of local anaesthesia).
  • amyloid deposition e.g. treatment of AD
  • nervous tissue repair e.g. treatment of spinal injury
  • myelination e.g. in the treatment of MS
  • stem cell function e.g. for brain repair
  • sodium channel activity e.g. treatment of epilepsy and/or induction of local anaesthesia
  • TAG-1 or sodium channel peptide complex proteins include but are not limited to Caspr2, NgR, K+ channels, TN-R, TN-C, MAG, Nogo-A, phosphacan, F3, OMgp, RPTP ⁇ and NG2.
  • drugs, peptides or other molecules include but are not limited to peptide fragments of or antibodies to Caspr, NgR, K+ channels, TN-R, TNB-C, MAG, Nogo, phosphacan, F3, OMgp, RPTP ⁇ and NG2.
  • the present invention also provides a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, a neurological condition in a subject, the method comprising: (a) providing at least one sample from a subject; (b) determining the expression and/or activity of amyloid precursor protein, its derivative or a fragment thereof; (c) comparing the expression and/or activity of the amyloid precursor protein, its derivative or a fragment thereof, with that of at least one control, a difference in expression and/or activity indicating the presence of, or predisposition to, and/or severity of a neurological condition in the subject.
  • the present invention also provides a method of monitoring the efficacy of a treatment for a neurological condition in a subject, the method comprising: (a) providing at least two samples from a subject, each sample obtained at different time points; (b) determining the expression or activity of amyloid precursor protein, its derivative or a fragment thereof; and (c) comparing the expression and/or activity of the amyloid precursor protein, its derivative or a fragment thereof, in the at least two samples.
  • the present invention also provides a method of prognosticating the outcome of a neurological condition in a subject, the method comprising: (a) providing at least one sample from a subject; (b) determining the expression and/or activity of amyloid precursor protein, its derivative or a fragment thereof; (c) comparing the expression and/or activity of the amyloid precursor protein, its derivative or a fragment thereof, with that of at least one control, a difference in expression and/or activity indicating the prognosis of a neurological condition in the subject.
  • the present invention also provides a method of inducing analgesia and/or anesthesia in a subject, the method comprising modulating the expression and/or activity of amyloid precursor protein, its derivative or a fragment thereof.
  • the present invention also provides a method of modulating differentiation of at least one stem cell, the method comprising decreasing expression and/or activity of amyloid precursor protein, its derivative or a fragment thereof, in the at least one stem cell.
  • the decreasing may comprise administering an effective amount of a compound selected from the group consisting of TAG-1 , anesthetics, toxins and Go protein(s).
  • conduction velocity measures as a marker for APP-sodium channel interactions and hence as a biomarker for APP malfunction and propensity for Alzheimer's disease or other amyloid-related dementia.
  • a method of detecting and/or quantitating the presence of, predisposition to, and/or severity of, at least one condition related to beta amyloid (A ⁇ ) deposition and/or sodium channel dysfunction in a subject comprising: (a) measuring the nerve and/or axon conduction velocity in the subject; (b) comparing the nerve and/or axon conduction velocity with that of at least one control, a difference in velocity indicating the presence of, or predisposition to, and/or severity of at least one condition related to A ⁇ deposition and/or sodium channel dysfunction in the subject.
  • the method may be for detecting and/or quantitating the presence of, predisposition to, and/or severity of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • the present invention also provides at least one pharmaceutical composition for treating at least one APP and/or sodium channel related medical condition comprising at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1 , a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s) toxin(s) and Go protein(s); and optionally at least one pharmaceutically acceptable diluent, carrier and/or excipient.
  • the medical condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis, brain injury, spinal cord injury, axonal injury-related disorders, epilepsy, neurodegenerative disorders and demyelination disorders.
  • the present invention also provides use of at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1, at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s) toxin(s) and Go protein(s), for the preparation of a medicament (or pharmaceutical composition) for the treatment of an APP and/or sodium channel related medical condition.
  • the pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier, diluent, adjuvant, excipients, or a combination thereof.
  • suitable excipients are water, saline, dextrose, glycerol, ethanol and the like as well as combinations thereof.
  • Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or alternatively the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carrier, excipient and/or diluent.
  • Excipients normally employed for such formulations includes mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the pharmaceutical composition may be for local, subcutaneous, intravenal, parenteral and/or oral administration.
  • the pharmaceutical composition may be administered through subcutaneous and/or intramuscular injection.
  • the pharmaceutical composition may be formulated as solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, aerosols, powders, or granulates.
  • the dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration.
  • the present invention also provides a kit comprising at least one compound selected from the group consisting of: APP, APP derivative, APP fragment, antibody to APP, TAG-1, at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Caspr, Caspr derivative, Caspr fragment, anesthetic(s) toxin(s) and Go protein(s), for the detection and/or diagnosis of at least one APP and/or sodium channel related medical condition and/or for the detection and/or diagnosis of at least one APP and/or sodium channel related medical condition and/or or treating and/or preventing at least one APP and/or sodium channel related medical condition.
  • APP APP derivative, APP fragment, antibody to APP, TAG-1, at least one TAG-1 complex protein, a TAG-1 homologous protein or fragment thereof, antibody to TAG-1 , sodium channel(s), at least one sodium channel complex protein, Cas
  • Rabbit polyclonal antibodies against Caspr (Nie et al, 2003) and monoclonal antibodies against TN-R (596, 619) (Xiao et al, 1998) and APP (Selkoe et al, 1988) were described previously. The antibodies recognise specifically APP intracellular domain (Selkoe et al., 1988).
  • Rabbit polyclonal anti-Nav1.6, Nav ⁇ 2 , pan-Nav (Alomone laboratories, Israel), monoclonal antibodies against pan-Na + channel (K58/35; Sigma), potassium channels Kv1.2 (K14/16) (Upstate, USA), and ⁇ -tubulin (GTU88; Sigma) were obtained from the respective commercial sources.
  • mice have been previously described.
  • APP knock-out mice exhibit decreased locomotor activity and forelimb grip strength
  • APP transgenic mice over-express human APP driven by the prion promoter.
  • PLP transgenic mouse (PLP tg; Inoue et al., 1996; Rasband et al., 2003) is an animal model of severe demyelination.
  • mice All experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of Singapore General Hospital.
  • IACUC Institutional Animal Care and Use Committee
  • Xenopus laevis oocytes were approved by the IACUC of the National University of Singapore.
  • IF labelling Animals were perfused transcardially with 0.1 M Ringer's solution and 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) sequentially. Spinal cords were removed immediately for different subsequent preparations. For cryosection, tissues were immersed, first in 15% sucrose and then in 30% sucrose in PB. Ten micrometer-thick sections were mounted onto gelatin or poly-l-lysine coated glass slides. After being air-dried, slides were kept at -2O 0 C for later use.
  • tissue slices were first immersed in absolute acetone at -20 0 C for 20 min. Slices were then thoroughly washed in 0.1 M phosphate buffered saline (PBS, pH 7.6) containing 0.3% Triton X-100. After blocking with 10% normal goat serum (NGS) diluted in PB for 1 hr, tissue slices were incubated with primary antibodies at their optimum dilutions, either overnight or 2 hrs at room temperature (RT). Cy2- or Cy3-conjugated secondary antibodies (Amersham) corresponding to the primary antibodies were used for further incubation at RT for 1 hr. For double labelling, one more cycle of labelling was similarly conducted for second primary antibodies. All labeled slices were visualized and photographed under a Leica RXA2 upright fluorescence microscope or an LSM5 Carl Zeiss confocal microscopic system.
  • Brain tissue or cells were homogenized in ice-cold homogenizing buffer (320 mM sucrose, 10 mM Tris-HCI pH 7.4, 1 mM NaHCO 3 pH 7.4, 1 mM MgCb) supplemented with 1% protease inhibitor cocktail (Amersham), and subsequently centrifuged at 5,000 g for 15 min. The supernatant was collected and spun at 60,000 g (Beckman ultracentrifuge) for 60 min at 4 0 C.
  • homogenizing buffer 320 mM sucrose, 10 mM Tris-HCI pH 7.4, 1 mM NaHCO 3 pH 7.4, 1 mM MgCb
  • Amersham 1% protease inhibitor cocktail
  • a lysis buffer (10 mM Tris-HCI pH9, 150 mM NaCI, 0.5% Triton X-100, 1% sodium deoxycholate (DOC), 0.5% SDS, 2 mM EDTA, and 1% protease inhibitor cocktail. All samples were subjected to Bradford protein assay.
  • Equal amounts of protein were separated on acrylamide gels and transferred onto nitrocellulose membranes.
  • Western blotting was performed under standard conditions, applying rabbit polyclonal antibodies against APP (1 :1000) and mouse monoclonal antibodies against ⁇ -tubulin (1 :1000). The latter protein was used for loading normalization. Either anti-mouse, or anti-rabbit peroxidase- conjugated secondary antibodies were applied at 1 :10,000 and blots were visualized with an ECLTM detection kit (Amersham).
  • Electromicroscopy Animals were transcardially perfusion-fixed with 2% paraformaldehyde - 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for electron microscopy.
  • Spinal cords and sciatic nerves were removed and post-fixed in the same fixative solution for 2 hrs. After being finely trimmed, the samples were immersion-washed overnight.
  • Tissue blocks were further post-fixed in 1% osmium tetroxide (OsO 4 ) containing 1.5% potassium ferrocyanide for 2 hrs, and thereafter subjected to dehydration in ascending series of alcohol and acetone. After undergoing gradual infiltration with Araldite 502 (EMS), the blocks were embedded and polymerized overnight at 60 0 C.
  • OsO 4 osmium tetroxide
  • the vector pD4GI_ a modified version of the pcDNA4A vector (Invitrogen) that contains the Xenopus ⁇ -globin untranslated regions, was used to boost expression in oocytes.
  • the Xenopus ⁇ -globin 5' and 3' untranslated regions were subcloned into the pcDNA4A vector.
  • cDNAs encoding the rat ⁇ 1 and ⁇ 2 subunits (provided by Lori Isom, University of Michigan), the AICD domain (SEQ ID NO: 4 cDNA; SEQ ID NO: 7,peptide), C-terminal fragment C31 domain (SEQ ID NO: 8, cDNA; SEQ ID NO: 10, peptide), G protein Go binding domain (SEQ ID NO: 11 ; SEQ ID NO: 14, peptide), Fe65 binding domain (SEQ ID NO: 15, cDNA; SEQ ID NO: 18, peptide) and a short peptide motif Val667- Arg672 (SEQ ID NO: 19 cDNA;SEQ ID NO:22, peptide) were inserted between these two regions.
  • a Kozak sequence was added to around the start codon (GCCACCATGG) (SEQ ID NO:1) for oocyte expression.
  • the pLCT1-Scn8a plasmid containing mouse Nav1.6 cDNA was provided by Dr. Alan L. Goldin (University of California, Irvine).
  • the plasmid containing cDNA of Nav1.5 ⁇ subunit of sodium chanenels was provided by Dr. A. L. George (Vanderbilt University, Nashville).
  • the plasmid encoding the human App695 cDNA was provided by Dr. Carsten Schmidt (University of Hamburg).
  • the AlCD domain (forward primer: 5'-TTTGGTG AAGCTT GCCACCATG GTG ATG CTG AAG AAG AAA CAG TAC-3' (SEQ ID NO: 5); reverse primer: 5'- GGCG GCGGCCGC CTA GTT CTG CAT CTG CTC AAA GA-3 1 (SEQ ID NO: 6)
  • G protein Go binding domain (forward primer: 5'-GCGC AAGCTT GCCACC ATG CATCATGGTGTGG TGGAGGTTG-3' (SEQ ID NO: 12); reverse primer: 5'- GGCG GCGGCCGC CTA CTT GGA CAG GTG GCG CTC CTC-3(SEQ ID NO: 13), C-terminal C31 domain (forward primer: 5'-GCGC AAGCTT GCCACC ATG GCCGCTGTCACCCCAGAGGAG-3' (SEQ ID NO: 9); reverse primer: 5'GGCG GCGGCCGC CTA GTTCTGCATCTGCTCAAAGA-3'(SEQ ID NO: 6), Fe
  • Plasmids were purified using QIAGEN Plasmid Maxi Kit (QIAGEN). All these purified constructs were linearized, and cRNAs were synthesized in vitro using the mMESSAGE mMACHINETM T7 cRNA synthesis kit (Ambion). The concentration of cRNA was determined from OD (260 nm).
  • Stage V oocytes were surgically removed from mature female Xenopus laevis
  • oocytes were placed in 96-well plates and incubated at 19 0 C in Barth's solution overnight.
  • oocytes were injected (Roboocyte, Multichannel Systems, Germany) with cRNAs.
  • RNA was dissolved in 1 mM Tris-HCI, pH 7.5.
  • the molar ratio of ⁇ to ⁇ subunits was 1 :10, while that of the ⁇ subunit to APP was 1 :1.
  • the oocytes were then incubated at 19°C.
  • Membrane currents were recorded from oocytes with the two-microelectrode voltage-clamp technique at room temperature (19°C to 21 0 C) 2 to 3 days after injection of cRNA.
  • Oocytes were held at -100 mV and 45 ms steps to command potentials between -75 mV and 40 mV were made in 5 mV increments to assess the current-voltage (I-V) relationship.
  • Electrophysiological recordings of dorsal root ganglion neurons Acutely isolated dorsal root ganglion (DRG) neurons were prepared from the sensorimotor cortex of anesthetized and then decapitated adult APP +/+ and APP 'A C57BL mice. The lumbar segments of vertebrate column were dissected. The DRGs, together with the nerve roots, were quickly removed and transferred immediately into Dulbecco's modified Eagle's Medium (DMEM, Gibco, Carlsbad, CA).
  • DMG Dulbecco's modified Eagle's Medium
  • the DRGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 1.5 ml of DMEM in which trypsin (type III, 0.5 mg/ml, Sigma, St. Louis, MO), collagenase (type I, 1 mg/ml, Sigma) and DNase (type I, 0.1 mg/ml, Sigma) had been dissolved. After incubation at 35°C in a shaking water bath for 30 min, soybean trypsin inhibitor (type ll-s, 1.25 mg/ml, Sigma) was then added to stop trypsin digestion.
  • trypsin type III, 0.5 mg/ml, Sigma, St. Louis, MO
  • collagenase type I, 1 mg/ml, Sigma
  • DNase type I, 0.1 mg/ml, Sigma
  • DRGs were carefully removed by Pasteur pipette and placed in bath solution containing (in mM): 140 NaCI, 3 KCI, 1 MgCI 2 , 1 CaCI 2 , 0.1 CdCI 2 , and 20 HEPES, pH 7.3 adjusted to 320 mosmol/l with glucose.
  • the DRGs were mechanically dispersed (5 strokes up and down) with a 1-ml pipette and plated onto a 35- mm culture dish and kept for at least 30 min before electrophysiological recordings. All recordings were made at room temperature (20-24 0 C) within 10 h after dissociation to keep the experiment as similar to in vivo as possible.
  • CdCb in bath solution was used to block Ca 2+ currents.
  • CsF concentration was decreased to 120 mM in the pipette solution.
  • the pipette potential was zeroed before seal formation, and the voltages were not corrected for liquid junction potential. Capacity transients were cancelled, and series resistance was compensated (75-80%) as necessary.
  • the leakage current was digitally subtracted on-line using hyperpolarizing control pulses, applied before the test pulse, of one-fourth test pulse amplitude (P/4 procedure). Data are presented as means ⁇ S. E and statistical analyses were performed using the Student's t test (significance at least p ⁇ 0.05).
  • I-V Standard current-voltage
  • Activation curves were fitted with the following Boltzmann distribution equation: G N a / - V m )/k], where G Na is the voltage-dependent sodium conductance, GNa.max is the maximal sodium conductance, V 1/2 is the potential at which activation is half- maximal, V m is the membrane potential, and k is the slope.
  • Availability protocols consisted of a series of pre-pulses (-100 to 1OmV) lasting 100 ms from the holding potential of -7OmV, followed by a 100 ms depolarization to -10 mV.
  • the animals were anesthetized, and then decapitated and the spinal cord extracted by rapid laminectomy and washed in oxygenated Krebs' solution (NaCI 119mM, KCI 2.5mM, NaH 2 PO 4 1mM, MgSO 4 1.3mM, CaCI 2 2.5mM, D-glucose 11mM, NaHCO 3 26.2mM, equilibrated with 95% O 2 , 5%CO 2 ).
  • the cord was incubated in Krebs' solution at room temperature for at least 1 hr, and then mounted in a recording chamber. The temperature of chamber was raised to 32 0 C and 37°C, and a series of recordings were made to stimulation that was adjusted to 10% above the level that elicited a maximum response.
  • the data were digitized at 20 kHz for subsequent analysis.
  • Stimulation was delivered onto one end of nerve by a concentric bipolar electrodes (Frederick Haer Co., Bowdoinham, ME, USA) and the other end of the nerve was drawn into a suction electrode for recording action potentials
  • Availability protocols consisted of a series of prepulses (-120 to +2OmV) lasting 500ms, from the holding potential of -9OmV, followed by a 40ms depolarization to -1OmV, every 10s.
  • Myelinated axons in the CNS are characterized by their segmental structures: the node of Ranvier (NOR), paranode, juxtaparanode, and internode.
  • NOR Ranvier
  • paranode paranode
  • juxtaparanode juxtaparanode
  • internode internode.
  • Myelination is a dynamic process mediated by bidirectional axoglial interactions that are involved in the clustering of axonal molecules, such as Na+ channels, F3/contactin, and OMgp, at NORs (Salzer 2003; Ma et al., 2007).
  • axonal molecules such as Na+ channels, F3/contactin, and OMgp
  • Fig. 19A Western blotting analysis
  • APP was expressed in brain as early as at birth (PO); however, it was greatly up-regulated between postnatal day 5 (P5) to day 21 (P21).
  • the intensity of APP nodal clustering was quantified in relation to Kv1.2 during development.
  • the ratio of APP to Kv1.2, reflecting the developmental progress of APP clustering in the population of axons, was 50% at P10, 60% at P15 and P21 , and 90% at 2 months (Fig. 7), implying that APP clustering also occurs latter than the K+ channel clustering in the CNS.
  • Example 3 nodal accumulation of APP relies on the integrity of distinct axonal domains
  • APP has long been utilized as a marker for axonal degeneration following neural injury (Kuhlmann et al, 2002).
  • APP co- localizes extensively with Nav1.6 and to a lesser extent with Nav1.2 sodium channels at sites of axonal injury. This is evidenced by diffuse distribution of both APP, Nav 1.6 and, to a lesser extent, Nav1.2 along demyelinated axons and an increase in the number of diffusely APP-positive axons (Craner et al, 2004). These observations suggest a spatial relationship between APP and these two nodal channels in this pathological condition.
  • PLP tg 7-month-old PLP transgenic mice
  • PLP tg mice double labelling of K+ channels (red) or the 200 kDa neurofilament (red), two axonal markers, with APP (green) revealed that APP clustering completely disappeared in demyelinated axons (Fig. 19B).
  • APP clustering along axons is severely disrupted in PLP tg mice as well as in the EAE model, these results support the notion that nodal accumulation of APP relies on the integrity of distinct axonal domains.
  • APP knock-out mice exhibit decreased locomotor activity and forelimb grip strength (Zheng et al, 1995), consistent with myelinated axon-related abnormalities.
  • EM electron microscopy
  • the g ratio (the numeric ratio between the diameter of the axon and the outer diameter of the myelinated fiber (Michailov, 2004) was significantly increased in the spinal cord of the knock-out mice 0.829 ⁇ 0.002) in comparison to wild-type mice (0.817 ⁇ 0.002) (Fig. 9c; p ⁇ 0.001).
  • axon diameter ⁇ 1.0 ⁇ m; 1.0-1.5 ⁇ m; 1.5-2.0 ⁇ m; 2.0-2.5 ⁇ m, 2.5-3.0 ⁇ m; 3.0-4.0 ⁇ m; and >4.0 ⁇ m).
  • the degree of peripheral myelination in the sciatic nerve from APP wild-type and knock-out mice was analysed by using EM.
  • wild-type mice the average g ratio was 0.715 ⁇ 0.002 whereas myelin in APP-/- mice was significantly thinner (p ⁇ 0.001) with a g ratio of 0.754 ⁇ 0.038 (Fig. 10), indicating a thinner myelin sheath encapsulating the axons.
  • the analysis of g ratios across all axon sizes was done.
  • Example 5 - APP plays a role in mvelination of axons
  • Example 6 - APP interacts with sodium channels
  • Voltage-gated Na+ channels consist of a pore-forming ⁇ subunit and at least one ⁇ subunit that regulates channel behavior and surface expression (Catterall, 2000).
  • Nav1.6 produces both transient and persistent currents (Rush et al, 2005) and localizes predominantly at adult NORs, but not in the intemodal region of myelinated axons (Caldwell, 2000).
  • Na+ channel ⁇ 1 and ⁇ 2 subunits also localize at NORs (Chen et al, 2004).
  • the inventors coexpressed Nav1.6 with full length APP in various molar ratios in Xenopus laevis oocytes to investigate whether APP could act as a ⁇ subunit of sodium channels. Notably, the increase in the sodium current in the presence of APP was not in a dose dependent manner, indicating that APP does not act as a ⁇ subunit of sodium channels (Fig. 28).
  • APP increased the peak current 1.74 ⁇ 0.11 fold on expression of the ⁇ subunit alone (FIG. 23a; p ⁇ 0.001), 2.38 ⁇ 0.55 fold on expression of the ⁇ subunit and ⁇ 1 subunit in combination (FIG. 23b; p ⁇ 0.001), 1.84 ⁇ 0.21 fold on expression of the ⁇ subunit and ⁇ 2 subunit in combination (FIG. 23c; p ⁇ 0.001), and 2.47 ⁇ 0.22 fold on expression of the ⁇ subunit together with both ⁇ 1 and ⁇ 2 subunits (FIG. 23d; p ⁇ 0.001).
  • the increase in the current in the presence of APP appeared to be greater when the ⁇ 1 subunit was expressed as the sole ⁇ subunit (Fig.
  • APP had no significant effect on activation when expressed together with the ⁇ subunit alone (Fig. 13c) or the ⁇ and ⁇ 2 subunits (Fig. 15c), but shifted V1/2 to the left when the ⁇ 1 subunit was expressed together with the ⁇ subunit (from -
  • Inactivation protocols consisted of a series of pre-pulses followed by a short depolarisation. The normalized curves were fitted using a Boltzmann distribution equation. The presence of APP had no significant effect on the Na+ inactivation currents (Fig. 13d, 14d, 15d, and 16d). For recovery from inactivation experiments, two pulses were applied with a variable recovery time (1 to 30 ms) at a holding potential of -100 mV. Curves were fitted with an exponential function. The presence of APP had no significant effect on the recovery from inactivation currents in the presence of the ⁇ subunit alone (Fig. 13e) or together with the ⁇ 1 subunit (Fig. 14e).
  • APP interacts both with the ⁇ subunit alone and with the ⁇ subunits.
  • Example 7 Whole cell recordings from DRG neurons
  • Na+ current densities were significantly decreased in DRG neurons from APP knockout mice versus wild-type mice (p ⁇ 0.05).
  • the activation curve of the Na+ conductance was constructed from the current-voltage relationship. There was no difference in the activation of Na+ currents in the wild type and APP-deficient mice (p > 0.05; Fig. 17b).
  • Steady-state inactivation of Na+ currents was determined. Current amplitudes were expressed as a fraction of the maximal current amplitude and plotted as a function of the prepulse potential. Steady- state inactivation did significantly differ between the wild-type and APP-deficient mice (Fig. 17c). Recovery from inactivation was studied by gradually increasing the interval (1 to 400ms) between two depolarising test pulses.
  • APP clustering only occurs in the CNS
  • the significant change of nerve conduction in the mutant spinal cord, but not sciatic nerve implies that the clustered APP is an important factor that plays a specific role in modulation of the CNS action potentials.
  • APP 1 its fragments and derivatives, and that of molecules, cellular components, drugs and/or antibodies that interact with APP, its fragments and derivatives, many biological and medical events, functions and conditions, particularly neurological conditions, may be treated.
  • Nav1.6 sodium channel ⁇ subunit is abnormally downregulated in the CNS of APP null mice.
  • Nav1.6 is the predominant_ ⁇ subunit of sodium channels that locate at CNS NORs (Salzer, 2003).
  • the present inventors ascertained as to whether there might be differences in the level of Nav1.6 expression between APF 1' mice and their wt littermates.
  • Western blot results showed that the expression of Nav1.6 was abnormally decreased in the brain in APF' ' mice versus APP +/+ and APP +/' littermates (Fig. 24).
  • the abnormal changes in locomotor activity, forelimb grip strength (Zheng et al., 1995), nerve conduction, and Nav1.6 expression in the APF 1' mice strongly imply that APP may act as a specific modulator of Nav1.6 at the CNS NORs.
  • Example 9 - VTPEER domain of APP increases sodium current expression in Xenopus oocytes expressing the Nav1.6 sodium channel ⁇ subunit.
  • the present inventors also determined which domain(s) in APP could involve in increment of sodium current expression in Xenopus oocytes expressing the Nav1.6 sodium channel a subunit.
  • APP intracellular domain AICD; X. Cao and S ⁇ dhof (2001)
  • Go protein-binding domain GB; H 657 -K 676 of APP-695; Nishimoto et al., 1993; Brouillet et al., 1999
  • Fe65 binding domain Fe65BD; v 666_ ⁇ 686 ; Ermekova et aL j ,, Q 97 J 1 and Q 31 domain ( C31 ; A 664_ N 695. L
  • VTPEER is a common overlapped peptide sequence in the AICD, GB, Fe65BD, and C31 .
  • the inventors further coexpressed this peptide with the Nav1.6 a subunit in Xenopus laevis oocytes.
  • VTPEER increased the peak sodium current as well (Fig. 26e; Figure 25). All the intracellular domains had no significant effect on activation, inactivation or recovery from inactivation (Figure 28).
  • VTPEER peptide mediates the effects of APP on sodium currents.
  • Example 10 - Go protein is involved in the modulation of sodium currents by APP.
  • APP intracellular portion contains a Go protein-binding (GB) domain, His 657 -Lys 676 of APP-695 (Nishimoto et al., 1993; Lang et al., 1995; Okamoto et al., 1995; Brouillet et al., 1999).
  • Go protein-binding (GB) domain His 657 -Lys 676 of APP-695
  • Go protein might mediate the APP- dependent modulation of sodium currents.
  • APP is clustered specifically at the NORs in the CNS, but not PNS. Consistently, conduction velocities are significantly impaired only in the CNS, but not PNS, in APP mutant mice. Moreover, APP positively modulates the function of sodium channels, increasing sodium 0 currents through its intracellular VTPEER peptide via a Go protein-coupled pathway.
  • APP is a novel component at the CNS NORs.
  • TAG-1/TAX from the same L1 cell adhesion molecule subfamily as F3/contactin, has been suggested to be a constituent of juxtaparanodal formation (Traka et al., 2002 and Poliak et al., 2003).
  • the sodium channel ⁇ subunits which associate with ⁇ subunits of voltage-gated sodium channels at the NORs, have an extracellular immunoglobulin-like loop and act as cell adhesion molecules (McEwen and Isom, 2004).
  • APP is expressed predominantly in axons and is upregulated on neural injury and has been utilized as a marker for axonal degeneration. The present work shows that APP is aggregated specifically at the NORs in the CNS, but not PNS.
  • APP clustering of APP occurs later than the clustering of sodium and potassium channels. This aggregation requires the integrity of distinct axonal domains as evidenced in both physiological and pathological conditions. Conversely, APP is not required for clustering of other nodal molecules such as tenascin-R, Caspr and sodium channels, as clustering of these molecules still occurs in APP null mice. APP clustering starts from P10 during myelination and disappears after demyelination in two animal models. Thus, nodal APP clustering in the CNS may be the most important factor related to the phenotype of reduced conduction velocities in the CNS, but not PNS, in APP mutant mice. However, the molecular mechanisms underlying the clustering of APP at CNS NORs remain to be explored.
  • the structure of the NORs is formed by the halt of lateral expansions of two apposing oligodendrocytes in the CNS.
  • the voltage-gated sodium channels are highly concentrated at this region with a density of 1 ,500 molecules/ ⁇ m 2 , but of less than 100 molecules/ ⁇ m 2 in the non-nodal areas (Rosenbluth 1999). Since the myelin sheath insulates the wrapped portion of the axon, the action potential is conducted in a saltatory manner, thus significantly increasing the conduction speed' Sodium channels are responsible for depolarizing and repolarizing events during transmission of the action potential along axons.
  • the fine modulation of sodium channels at the NORs is essential for precise saltatory conduction along myelinated axons.
  • sodium currents are positively modulated by axonal molecules, such as F3/contactin, ⁇ subunits of sodium channels, and glia-derived molecules, such as tenascin-R and -C (Kazarinova-Noyes et al., (2001); lsom et al., (1995); Srinivasan et al., (1998); and Ma et al., (2007).
  • APP does not act as a ⁇ subunit of sodium channels, as the increase in the sodium current in the presence of APP was not in a dose dependent manner (Figure 29).
  • APP clusters at CNS NORs and increases Nav1.6 sodium currents in oocytes.
  • the APP-induced increase in the sodium currents is mediated by its intracellular VTPEER peptide, a common domain shared by AICD, GB, Fe65BD, and C31. Consistent with the augmentation of sodium currents by APP, spinal conduction velocities are impaired in APP null mice.
  • the APP-induced increase in the sodium currents is also mediated by the His 657 -Lys 676 Go protein binding domain in the AICD of APP-695 and appears to involve the heterotrimeric G protein Go.
  • This G protein binding domain is known to selectively activate the heterotrimeric G protein Go with which it complexes (Nishimoto et al., 1993; Brouillet et al., 1999).
  • APP modulation of Nav1.6 via G protein has not previously been reported, our findings are consistent with G protein-mediated modulation of various other sodium channels ( Komwatana et al., 1996; Ma et al., 1997; Lu et al., 1999).
  • a ⁇ amyloid
  • Deposition of A ⁇ is considered to be a marker of the pathology of AD and is associated with other pathological markers of AD such as cell death in the CNS, accumulation of amyloid plaques, and the appearance of neurofibrillary tangles.
  • APP is enriched in presynaptic terminals (Ferreira et al 1993). Although the release of component peptides of APP, including A ⁇ deposition, has been thought to occur predominantly at synapses (Ferreira et al (1993), increasing evidence strongly suggests that an important component of AD is damage to myelin.
  • the entorhinal cortex from where the perforant pathway originates, is one of the most severely affected areas, particularly at earlier stages of the disease. Moreover, electrical activity within the pathway modulates interstitial fluid A ⁇ levels, which can be blocked by tetrodotoxin (TTX), a specific sodium channel blocker (Cirrito et al., 2005).
  • TTX tetrodotoxin
  • NORs are the naked regions of the axolemma and intermittently distribute along myelinated axons, implying potential interactions between exposed axonal molecules and the extracellular partners.
  • TAG-1 acts as a functional ligand for APP to trigger AICD transcriptional activation and negatively modulate neurogenesis
  • TAG-1 contactin-2
  • F3/contactin family of glycolphosphatidylinositol-linked immunoglobulin superfamily adhesion molecules a member of the F3/contactin family of glycolphosphatidylinositol-linked immunoglobulin superfamily adhesion molecules.
  • Cell adhesion assays and biochemical approaches established that TAG-1 and APP interact to trigger AICD's transcriptional activity in a ⁇ -secretase-dependent manner.
  • both APP and TAG-1 were shown to co-localize in the neural stem cell niche of the fetal ventricular zone and in addition, neurogenesis is enhanced in neural precursor cells isolated from TAG-1 -null, APP-null and TAG-1 /APP double knock-out mice.
  • TAG-1 the human homolog of TAG-1
  • TAX the human homolog of TAG-1
  • transfected CHO cells were seeded onto the culture dish dotted with APP-Fc
  • cells readily adhered to the APP protein spot (Fig. 30Aa and Fig. 30B), indicating that APP binds to TAG-1 or TAX.
  • This adhesion could be blocked by pre-treating the cells with anti-TAG-1 antibody (Fig. 30Ab, Fig. 30B) or the culture dish with anti-APP antibody (22C11 ; Fig. 30Ac, Fig. 30B), indicating that the interaction of TAG-1 with APP contributes to the adhesion.
  • the reciprocal adhesion assays were performed by plating APP-transfected CHO cells onto culture dishes with TAG-1-Fc protein spots. Similar adhesion of the cells to the protein spot were observed (Fig. 30Af and Fig. 30B), and the adhesion was blocked by neutralization of coated TAG-1 protein spots (Fig. 30Ag and Fig. 30B) or cell membrane-bound APP (Fig. 30Ah and Fig. 30B) with their respective antibodies. Similarly, control non-transfected CHO cells did not adhere to the TAG-1 protein spot (Fig. 30Ai and Fig. 30B).
  • TAXIg-GST 6 immunoglobulin Ig domains
  • FNIII fibronectin type III repeats
  • APP-transfected CHO cells The cells bound to spots of both proteins (Fig. 28Aj and i), indicating that APP has at least two binding sites located in the TAG-1 Ig domains and FNIII repeats. Consistently, control non-transfected CHO cells adhered to neither these two proteins (Fig. 30Ak and m) nor GST (Fig. 30An).
  • AICD release and transcriptional activity The activation of AICD release and transcriptional activity is thought to be triggered by external cues.
  • ligands for the extracellular portion of APP that affects its proteolytic processing and signalling cascade have not been functionally characterised.
  • a previously reported luciferase reporter system where Gal4 is fused to the C- terminal of APP was introduced into CHO and L1-, or TAG-1- or TAX-transfected CHO cells._A significant upregulation of the reporter activity was observed in both TAG-1- and TAX-transfected CHO cells, but not in the control CHO cells as well as L1 -transfected CHO cells (Fig. 32a).
  • a specific ⁇ -secretase inhibitor reduced the TAG-1 triggered AICD transcriptional activity in a dose-dependent manner (Fig. 32b). That AICD luciferase activity is significantly increased by transfection with APP in both TAG and TAX transfected CHO cells suggest that the interaction between APP and TAG-1 is a critical step in triggering AICD activated transcription.
  • TAG-1 amd APP was studied by immunofluorescence (IF) labelling using antibodies against TAG-1 (a polyclonal anti-TAG-1) and APP (C7).
  • IF immunofluorescence
  • APP Fig. 33b, b ⁇ c and c'
  • TAG-1 Fig. 33e, e', f and f
  • nestin Fig. 33a and d
  • a neural progenitor marker a neural progenitor marker
  • LV a neural progenitor marker
  • double IF showed that APP (Fig. 33h and h') and TAG-1 (Fig. 33g and g') were co-expressed (Fig. 33i and i') in the LV walls.
  • NPCs from E14 mouse telencephalic ventricular walls were isolated. These cells were double-stained for APP (Fig. 34a' and c') or TAG-1 (Fig. 34b' and d') and the neural progenitor markers nestin (Fig. 34a and b) or Sox2 (Fig. 34c and d). The results suggest that both APP and TAG-1 are expressed by the NPCs.
  • TAG-1 /APP signaling pathway in modulation of neurogenesis TAG-1 /APP signaling pathway in modulation of neurogenesis.
  • NPCs were isolated from the E14 telencephalic ventricular walls of APP-/- (Fig. 35A) and TAG-1-/- (Fig. 35B) mice, respectively.
  • TUJ1 green; Fig. 35Aa and b; 35Ba and b
  • MAP2 two markers for mature neurons (not shown), and DAPI (blue; Fig. 35Aa and b; Ca and b).
  • Both TUJ1- and MAP2-positive cells are significantly increased in APP (about 40%; Fig. 35Bc and d) and TAG-1 (about 45%; Fig. 35Bc and d) deficient mice compared with wild-type littermates.
  • the inventors generated TAG-1 -/-/APP-/- mice to confirm that TAG-1 /APP signaling plays a negative role in modulating neurogenesis.
  • TAG-1 -/- /APP-/- fetal NPCs the inventors studied whether AICD transcriptional activity could be also modulated by the TAG-1 /APP interaction.
  • TAG-1 -/-/APP-/- NPCs were cultured as monolayers loaded onto culture dishes co-coated with TAG-1 -Fc, F3-Fc, or L1- Fc and laminin.
  • TAG-1 -Fc but not F3-Fc, LI-Fc, and laminin, strongly triggered AICD reporter activity (Fig. 36Aa).
  • a specific ⁇ -secretase inhibitor blocked the induction of AICD transcriptional activity by TAG-1 in a dose-dependent manner (Fig. 36Ab), indicating that the ⁇ -secretase is involved in the TAG-1 triggered RIP process in the TAG-1 -/-/APP-/- fetal NPCs.
  • Mouse APP 695 cDNA encoding for the neuronal isoform of APP was subcloned into the pblue Bac vector using the BamHI and Sacl restriction sites.
  • APP-Fc primers for the Sacl restriction site at the 5 ' end (CTGACGGAACCAAGACCACCG; SEQ ID NO: 23) and for the COOH terminal end of the APP extracellular domain (terminating at amino acid position 624; SWISS-Prot accession number P12023) at the 3 ' end (GCTGAAGATGTGGGTTCGAACAAA; SEQ ID NO: 24) were used, introducing a new BcII restriction site at the 3 ' end.
  • This vector was used to stably transfect CHO K1 cells according to published procedures (Chen et al 1999).
  • TAG-1 -Fc recombinant protein A soluble form of TAG-1 -Fc recombinant protein was produced in 293T cells.
  • the signal sequence of the GPI-anchor of mouse TAG-1 was substituted with human IgG-Fc followed by a termination codon.
  • the recombinant cDNA was inserted at the Hind Ill-Not I sites of pDX, a modified pcDNA3 vector with an amplification-promoting sequence (APS) upstream of the CMV site (Hemann et al 1994).
  • the vector was introduced into 293T cells.
  • Fc proteins were purified via the Fc-tag using Protein-A column chromatography as described (Chen et al 1999).
  • the sequences of Ig and FNIII domains of TAG-1 were inserted into the pGEXKG vector to produce the GST-tagged fusion proteins.
  • the recombinant vectors were introduced into the TOP10 strain of E. coli, which was subsequently induced by IPTG (Bio-Rad).
  • the recombinant GST fusion proteins were purified by using GST beads (Sigma) as instructed by the manufacturer.
  • CHO cells were stably transfected with pcDNA 3.1 (-) containing the human cDNA sequence of APP 695, the predominantly neuronal expressed isoform of APP.
  • APP, F3, TAX, TAG-1 and mock transfected CHO cells were cultured in DMEM containing 10% fetal calf serum.
  • 35mm tissue culture petri dishes (Becton Dickinson) were coated with methanol solubilized nitrocellulose and then with proteins (12 ⁇ M) for 2 hours at 37 0 C in a humidified atmosphere. Subsequently, the dishes were washed and blocked overnight with 2% heat- inactivated fatty acid-free BSA (Sigma).
  • TAG-1 - transfected CHO or APP-transfected CHO were plated in 2 ml of DMEM (Gibco) containing 10% fetal calf serum at a density of 1.5 x 106cells/ml.
  • DMEM Gibco
  • the cells were gently washed and fixed with 2.5% glutaraldehyde and stained with 0.5% toluidine blue (Sigma) in 2.5% sodium carbonate. Blockage of adhesion was carried out using polyclonal anti-TAG-1 (1 :100) or anti-APP (1 :100) antibodies for 0.5 h pre-incubation. Cells adhering to the various spots were photographed and counted. The results were analyzed by Newman-Keuls test with p ⁇ 0.05 being considered significant.
  • Freshly prepared cerebral hemispheres of adult rats were harvested and solubilized in 2% Triton X-100.
  • the buffer homogenate was centrifuged at 13,00Og for 1 hour at 4 0 C and the supernatant was incubated for 45 min at room temperature with the glutathioneagarose or protein A-coupled agarose beads that had been incubated with TAG-1-Fc or APP-Fc. After washing the beads, proteins were eluted with SDS-PAGE sample buffer and immunoblotted with anti-APP or anti-TAG-1 antibodies, respectively.
  • mice brains were lysed with RIPA buffer containing protease inhibitor cocktail (Roche).
  • lysates were precleared with protein A- coupled agarose beads for 1 hrs and incubated with APP or TAG-1 antibodies together with protein A-coupled agarose for overnight at 4 0 C. Samples were washed with washing buffer before the beads were re-suspend in SDS buffer and boiled for 3-5 mins. Samples were subjected to SDSPAGE.Westem blot analysis was performed and developed with ECL reagents (Amersham).
  • the APP-Gal4 assay system has been previously described15.
  • L1-, TAG-1-, TAXtransfected CHO cells as well as CHO cells were cotransfected with the following plasmids (i) pG5E1B-luc (Gal4 reporter plasmid, 0.1 ⁇ g DNA); (ii) pCMV-LacZ ( ⁇ -galactosidase control plasmid, 0.05 ⁇ g DNA); (iii) pMstAPP
  • ⁇ -secretase inhibitor L-685, 458; Calbiochem
  • DMSO DMSO
  • the ⁇ -galactosidase expression plasmid pCMV/ ⁇ -Gal was included to monitor the transfection efficiency. Cells were lysed at 24 hours after transfection and assayed using the Steady-Glo Luciferase Assay Kit (Promega).
  • APP "7" mice 26 and TAG-T' " mice27 have been described previously.
  • APP/TAG- 1 doubly deficient mice were generated by intercrossing APP homozygous and TAG-1 homozygous (APP " ' " X TAG-1 "7” ). Animals heterozygous for both loci were intercrossed to each other (TAG-1 +/ 7APP+/- X TAG-1 +/ 7APP +/” ) to generate APP and TAG-1 doubly deficient (TAG-1 -/-/APP-/-) offspring.
  • DMEM/F12 Gibco
  • N2 supplement 20ng/ml bFGF
  • EGF 20ng/ml EGF
  • second passage neurospheres were collected and dissociated into single cells.
  • the cell suspension was seeded into 24-well dishes with 20,000 cells per well and was induced in DMEM/F12 culture medium containing N2 and 0.5% fetal calf serum for 7-8 days. lmmunocytochemistry and quantitation
  • the WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled. J Biol. Chem. 72(52), 32869-77 (1997). Ferreira, A., Caceres, A., & Kosik, K. S. lntraneuronal compartments of the amyloid precursor protein. J. Neurosci. 13, 3112-3123 (1993). Golde, T. E., Estus, S., Usiak, M., Younkin, L. H., & Younkin, S. G. Expression of beta amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR.
  • Kang, J. et al. The precursos of Alzheimer's disease amuloid A4 protein resembles a cell-surface receptor. Nature 325, 733-736 (1987).
  • Beta amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc. Natl. Acad. Sci. USA 85, 7341-7345 (1988).
  • the neuronal adhesion protein 5 TAG-1 is expressed by Schwann cells and oligodendrocytes and is localized to the juxtaparanodal region of myelinated fibers. J. Neurosci., 22, 3016-3024 (2002)

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Abstract

La présente invention concerne des procédés destinés à moduler des activités (fonctions), des événements et/ou des affections biologiques et/ou médicales. Les activités, événements ou affections biologiques ou médicales peuvent inclure une prédisposition à des affections neurologiques, la myélinisation et la démyélinisation de neurones et/ou la différenciation de cellules souches neurales, ainsi que leur début et leur progression. La présente invention concerne, en particulier, un ou plusieurs procédés destinés à moduler l'expression et/ou l'activité du précurseur du peptide bêta-amyloïde (APP) et/ou de ses dérivés ou fragments. La présente invention concerne également des procédés destinés à moduler l'expression et/ou l'activité de molécules, comme des canaux sodiques et/ou TAG-1, qui entrent en interaction avec l'APP et ses dérivés ou fragments.
PCT/SG2007/000283 2006-08-31 2007-08-29 Modulation de l'activité et/ou affection neurales Ceased WO2008027017A1 (fr)

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US9133437B2 (en) 2009-11-02 2015-09-15 Toagosei Co. Ltd. Cell proliferation-promoting peptide and use thereof
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WO2018060712A1 (fr) * 2016-09-29 2018-04-05 Oxford University Innovation Limited Méthode de traitement ou de prévention de la douleur ou d'une activité neuronale excessive ou de l'épilepsie
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