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WO2000067016A9 - Procedes d'identification de composes pour le traitement de la maladie d'alzheimer - Google Patents

Procedes d'identification de composes pour le traitement de la maladie d'alzheimer

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Publication number
WO2000067016A9
WO2000067016A9 PCT/US2000/011401 US0011401W WO0067016A9 WO 2000067016 A9 WO2000067016 A9 WO 2000067016A9 US 0011401 W US0011401 W US 0011401W WO 0067016 A9 WO0067016 A9 WO 0067016A9
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WIPO (PCT)
Prior art keywords
cell
compound
candidate compound
activity
mouse
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2000/011401
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English (en)
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WO2000067016A1 (fr
Inventor
Ralph A Nixon
Anne M Cataldo
Paul M Mathews
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NATHAN S KLINE INSTITUTE FOR PSYCHIATRIC RESEARCH
NATHAN S KLINE INST FOR PSYCHI
Original Assignee
NATHAN S KLINE INSTITUTE FOR PSYCHIATRIC RESEARCH
NATHAN S KLINE INST FOR PSYCHI
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Application filed by NATHAN S KLINE INSTITUTE FOR PSYCHIATRIC RESEARCH, NATHAN S KLINE INST FOR PSYCHI filed Critical NATHAN S KLINE INSTITUTE FOR PSYCHIATRIC RESEARCH
Priority to JP2000615804A priority Critical patent/JP2002543425A/ja
Priority to AU46734/00A priority patent/AU772054B2/en
Priority to EP00928500A priority patent/EP1181550A4/fr
Priority to CA002370177A priority patent/CA2370177A1/fr
Publication of WO2000067016A1 publication Critical patent/WO2000067016A1/fr
Anticipated expiration legal-status Critical
Publication of WO2000067016A9 publication Critical patent/WO2000067016A9/fr
Ceased legal-status Critical Current

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    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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    • 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
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    • A01K2267/0312—Animal model for Alzheimer's disease
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    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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    • C07—ORGANIC CHEMISTRY
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    • G01N2333/914—Hydrolases (3)
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to methods for identifying compounds useful in the treatment of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • EP neuronal endocytic pathway
  • APP amyloid precursor protein
  • ApoE apolipoprotein E
  • a ⁇ amyloid ⁇ peptide
  • Individual neuronal endosomes can be as much as 32-fold larger in volume than the normal average and the total endosome volume can be 3 -fold higher in SAD brain.
  • LS lysosomal acid hydrolases (including proteases) are targetted to early endosomes in increased amounts.
  • AD Despite the extensive study of AD, the pathogenetic significance of the upregulation of the EP remains unclear. Thus, there is also a long-sought need to understand the importance of increased endocytosis and lysosomal activity in AD.
  • the invention features methods for identifying compounds for the treatment of AD.
  • the invention features a method for identifying a candidate compound as a compound that is useful for the treatment of AD that includes: a) providing a Tn65Dn mouse; b) administering the candidate compound to the mouse; and c) measuring the abnormal activity of the EP and/or a change in A ⁇ generation.
  • a decrease in the EP activity compared to the activity in a control Tn65Dn mouse not contacted with the candidate compound identifies the candidate compound as a compound that is useful for the treatment of AD.
  • a decrease in A ⁇ generation in a mouse, relative to a mouse not contacted with the candidate compound also identifies a compound as one that is useful for the treatment of AD.
  • the invention features a method for identifying a candidate compound as a compound that is useful for the treatment of AD.
  • the method includes: a) providing a cell from a Tn65Dn mouse; b) contacting the cell with the candidate compound; and c) measuring the abnormal activity of the EP, wherein a decrease in the EP activity, compared to the activity in a control cell (also from a Tn65Dn mouse) not contacted with the candidate compound, identifies the candidate compound as a compound that is useful for the treatment of AD.
  • a ⁇ generation can also be used as a measurement; a decrease in A ⁇ generation in a cell mouse, relative to the control cell, also identifies a compound as one that is useful for the treatment of AD.
  • the cell is from a cell line derived from the mouse.
  • a preferred cell line includes a fibroblast cell line, a neuronal cell line, or a neuroblastoma cell line.
  • the cell is a fibroblast, a neuron, or an endothelial cell.
  • the invention features a method for identifying a candidate compound as a compound that is useful for the treatment of AD.
  • the method includes: a) providing a cell expressing a recombinant nucleic acid that causes abnormal activity of the endocytic pathway; b) contacting the cell with the candidate compound; and c) measuring the activity of the EP or A ⁇ generation activity, wherein a decrease in the abnormal activity, relative to the activity in a control cell expressing the recombinant nucleic acid but not contacted with the candidate compound, identifies the candidate compound as a compound that is useful for the treatment of AD.
  • the recombinant nucleic acid includes rab5, 46 kDa mannose 6-phosphate receptor (MPR46), cathespin (Cat D), or nucleic acids encoding other lysosomal hydrolases trafficked by the mannose 6- phosphate tag.
  • the cell is from a fibroblast cell line, a neuronal cell line, or a neuroblastoma cell line or is a fibroblast, a neuron, or an endothelial cell.
  • the cell is in an animal, or the cell is in vitro.
  • the invention features a method for identifying a candidate compound as a compound that is useful for the treatment of AD.
  • the method includes a) providing a mouse expressing a transgene that results in increased activity of the EP; b) administering the candidate compound to the mouse; and c) measuring the activity of the EP or A ⁇ formation activity, wherein a change in the abnormal activity, relative to the activity in a mouse expressing the transgene but not contacted with the candidate compound, identifies the candidate compound as a compound that is useful for the treatment of Alzheimer's disease.
  • the recombinant nucleic acid includes rab5, MPR46, Cat D, or other lysosomal hydrolases trafficked by the mannose 6- phosphate tag.
  • the abnormal activity includes increased endosomal fusion, increased endosomal recycling, upregulation of MPR46, accumulation of lysosomal hydrolases in early endosomes, or accumulation of A ⁇ in early endosomes.
  • the invention features a method for identifying a candidate compound as a compound that is useful for the treatment of Alzheimer's disease. This method includes the steps of: (a) providing a non- human animal; (b) administering to the animal a compound that induces lysosomal dysfunction; (c) administering to the animal a candidate compound; and (d) measuring neurodegeneration in the animal. A decrease in neurodegeneration, relative to an animal administered the compound that induces lysosomal dysfunction but not the candidate compound, identifies the candidate compound as a compound that is useful for the treatment of
  • the animal is a Tn65Dn mouse or a mouse expressing a transgene comprising a recombinant nucleic acid that increases activity of the endocytic pathway (such as the ones described herein).
  • the compound that induces lysosomal dysfunction is a lysosomal protease inhibitor, the animal has increased endocytic pathway activity relative to a normal animal.
  • abnormal activity of the endocytic pathway is meant an activity that is normally not observed in a control cell or animal.
  • Abnormal activities of the EP include: (i) increased endocytic rates; (ii) increased endosomal fusion and recycling; (iii) accumulation of lysosomal hydrolases in early endosomes; (iv) increased activity of lysosomal hydrolase in early endosomes; (v) upregulation of MPR46; and (vi) accumulation of ⁇ -amyloidogenic fragments in early endosomes.
  • a compound that "decreases” or “reduces” the abnormal activity of the endocytic pathway is one that exhibits any dimunition in one or more of the foregoing activities.
  • the decrease in abnormal activity is preferably by at least 5%, more preferably by at least 10%, and most preferably by at least 25%, 50% or more.
  • the percent change is usually measured for a period of hours or days, but can be measure in terms of weeks or even longer.
  • lysosomal dysfunction is meant an alteration in one function of the lysosomal system.
  • the alteration is preferably by at least 5%, more preferably by at least 10%, and most preferably by at least 25%, 50% or more.
  • the invention provides methods for identifying drugs useful for the treatment or prevention of AD. Additionally, the invention provides new drug targets for rational drug design.
  • Figs. 1A-1C are a series of photographs of tissue sections of prefrontal cortex of a control (Fig. IA) and two early stage AD cases (Figs. IB and IC) labeled with anti-rab5 antibody.
  • Figs. ID and IE are photographs of adjacent tissue sections of prefrontal cortex from an early stage AD case processed for A ⁇ immunohistochemistry (Fig. ID) or stained by Bielschowsky stain (Fig. IE).
  • Fig. IF is a graph showing that the average endosomal volume per neuron in the early stage AD cases was elevated compared to that of normal controls (control mean, 1.88%; possible AD mean, 3.68%; SAD mean, 5.04%).
  • Figs. 2A-2E are a series of photographs of tissue sections of prefrontal cortices from a fetus (Fig. 2A), infant (Fig. 2B-2D), or young child (Fig. 2E) with DS immunolabeled for rab5.
  • Figs. 3A and 3B are a series of photographs of tissue sections of the prefrontal cortex from early stage AD brains carrying the APOE ⁇ 4 genotype immunolabeled for rab5.
  • Fig. 3C is a schematic illustration showing that the endosomal volume per neuron was 50% higher in individuals carrying one or both copies of APOE ⁇ 4.
  • Figs. 4A-4E are a series of photographs showing early endosomal abnormalities in the cerebral vascular epithelium in heritable Dutch CAA (Figs. 4A-4C) and late onset FAD carrying the APOE ⁇ 4 allele (Fig. 4B).
  • Figs. 5A-5D are a series of photographs of immunofluorescence labeling of human MPR46 in control murine L cells (Fig. 5 A), or L cells stably- transfected with human MPR46 (Fig. 5B), MPR-HA (Fig. 5C), or MPR-HAY (Fig. 5D).
  • Figs. 5E and 5F are photographs of control L cells (Fig. 5E) or L cells stably-transfected with MPR-HAY (Fig. 5F) showing that MPR-HAY- transfected cells preferentially took up BODIPY-pepstatin from the medium.
  • Fig. 5G and 5H are photographs of immunofluorescence labeling of human MPR in N2a cells expressing MPR46 (Fig. 5G) or MPR-HAY (Fig. 5H).
  • Fig. 51 is a schematic illustration showing overexpression of MPR46 or a MPR46 trafficking mutant results in increased A ⁇ levels in the culture medium in cells overexpressing human APP.
  • Fig. 5J is a schematic illustration showing overexpression of MPR46 trafficking mutant results in increased A ⁇ levels in the culture media of cells that are not overexpressing APP.
  • Fig. 6A is a photograph of a Western blot showing rab5 immunoreactivity in control L cells and L cells stably-transfected with rab5.
  • Fig. 6B is a photograph of L cells stably-transfected with rab5Q 79 L showing enlarged endosomes (arrow).
  • Figs. 6C and 6D are photographs of control L cells (Fig. 6C) or L cells overexpressing wild-type rab5 (Fig. 6D) showing increased intemalization of Cy3 -labeled transferrin.
  • Fig. 7A is a photograph of a DNA agarose gel identifying 2 of 11 pups within a litter as carrying the Thy 1.1: MPR46 transgene.
  • Fig. 7B is a schematic illustration showing increased A ⁇ levels in the brain of a transgenic mouse expressing human MPR46 under the Thy 1.1 promoter.
  • Figs. 8 A and 8B are photographs showing rab5 immunoreactivity in cortical neurons from control mice (Fig. 8A) and mice with segmental trisomy 16 (Ts65Dn; Fig. 8B).
  • Figs. 8C -8F are photographs showing rab5 immunoreactivity in cortical neurons, particularly showing more labeling in Ts65Dn mice (Fig. 8F) compared to control mice (Fig. 8E).
  • Figs. 9A-C are photographs showing intracellular A ⁇ only in neurons of Ts65 on mice using antibody 4G8 (Fig. 9B) and an A ⁇ 40 specific antibody (Fig. 9C) but not in neurons of a control mouse (Fig. 9A).
  • Figs. 9D-F are photographs of immunofluorescence labeling showing the coincidence (Fig. 9F) of rab5 (Fig. 9D) and A ⁇ (Fig. 9E) in Ts65Dn neurons.
  • Fig. 10 is a schematic illustration showing increased levels of A ⁇ in the brains of Ts65Dn mice vs. control mice.
  • Figs. 11 A and 1 IB are photographs showing lipotuscin autofluorescence in sections of leupeptin-treated but not vehicle-treated brains.
  • Figs. I IC and 1 ID are photographs showing increased Cat D immunolabeling in sections of leupeptin-treated vs. vehicle-treated brains.
  • Fig. 1 IE is a photograph of a Western blot showing increased Cat D protein levels in the brain of leupeptin-treated mice when compared to control mice.
  • Fig. 1 IF is a schematic illustration showing increased Cat D activity in the brains of leupeptin-treated mice when compared to control mice.
  • Fig. 12 is a photograph of a Western blot showing an increase in the lower molecular weight forms of tau in leupeptin-treated mice.
  • Figs. 13 A and 13B are photographs of Cat D immunoreactivity in the brains of normal mice infused with vehicle (Fig. 13 A) or leupeptin (Fig. 13B).
  • Figs. 13C and 13D are photographs of Cat D immunoreactivity in the brains of Ts65Dn mice infused with vehicle (Fig. 13C) or leupeptin (Fig. 13D).
  • Figs. 14A-14D are photographs of Nissl staining of brain sections of a non-transgenic mouse infused for 4 weeks with leupeptin (Figs. 14A and 14C) and of a PS MI46L /APP swe transgenic mouse infused for 4 weeks with leupeptin (Figs. 14B and 14D).
  • Figs. 15A-15C are photomicrographs at low modification showing Nissl staining the extent of the cortical mantle in a non-transgenic mouse infused with vehicle (Fig. 15A), a non-transgenic mouse infused with leupeptin (Fig. 15B), and a PS M146L /APPswe transgenic mouse infused with leupeptin (Fig. 15C).
  • transgenic mice and transformed cell lines serve as models for understanding the cell biology underlying AD, as well as for testing candidate drugs for their efficacy and safety in the treatment of this prevalent disease.
  • Example 1 Endosomal abnormalities precede ⁇ -amyloid deposition and early neurofibrillary change
  • PS mutations do not alter endosomal function is consistent with the hypothesis that AD caused by PS1 mutations may occur via secretory pathway organelles (e.g., endoplasmic reticulum and the Golgi apparatus) rather than the EP.
  • LS activation is accentuated in PS-FAD, including in neuronal populations that are less vulnerable in SAD (e.g., laminae II and IV of prefrontal cortex).
  • Example 2 Accentuation of early endosomal abnormalities in individuals carrying the APOE ⁇ 4 allelle
  • tissue sections from a total of 15 early stage cases, consisting of 10 cases with APOE ⁇ 3 or APOE ⁇ 2 alleles and 5 cases carrying one or both copies of the APOE ⁇ 4 allele. These brains exhibited minimal histopathology and met the CERAD criteria of "possible AD.”
  • Neocortical sections were immunostained with rab5 antiserum and morphometric analyses were performed on groups of 25 neurons per brain. We found that the average mean endosomal volume/neuron in the early stage cases with the ⁇ 4 alleles was 50% larger than the AD cases with the ⁇ 3 or ⁇ 2 alleles (Fig.
  • Example 3 Transfected cell models of endosomal and hydrolase trafficking abnormalities in AD brain
  • MPR46 To mimic the hydrolase mistrafficking seen in AD, we isolated a cDNA encoding the human MPR46 and expressed this in stably transfected murine fibroblast-like cell lines (L cell; Fig. 5). In addition, we constructed two targeting mutants of MPR46 by replacing its C-terminal tail with that of the plasma membrane influenza virus hemagglutinin (MPR-HA) or a modified HA tail containing a tyrosine-residue endocytosis motif (MPR-HAY; Roth et al., J. Cell Biol. 102:1271-1283).
  • MPR-HA plasma membrane influenza virus hemagglutinin
  • MPR-HAY a modified HA tail containing a tyrosine-residue endocytosis motif
  • the wild-type MPR46 predominantly localized to perinuclear vesicles consistent with the trans-Golgi sacs and late endosomes (Fig. 5).
  • MPR-HA was expressed at the cell surface
  • MPR- HAY was localized primarily to small vesicles, which we have shown to be early endosomes by both rab5 immunolabeling and transferrin uptake.
  • Using a fluorescent-tagged pepstatin, an inhibitor of Cat D that binds to the proteolytically active enzyme we demonstrated that expression of the MPR- HAY chimera partially distributes this lysosomal hydrolase to early endosomes (Fig. 5).
  • a murine L cell line overexpressing APP was transfected with MPR46 (APP/MPR), a plasma membrane-targeted MPR46
  • APP/MPRHAwt a MPR46 construct that is preferentially trafficked to the early endosome
  • APP/MPRHAY a MPR46 construct that is preferentially trafficked to the early endosome
  • Transgenic mouse lines overexpressing any of the foregoing constructs provide useful animal models for identifying drugs useful for the treatment of AD. These mice can be made using standard techniques. If desired, the expression of the transgenes can be restricted to neurons by use of a promoter such as the Thy 1.1, neuron-specific enolase, or T ⁇ l ⁇ -tubulin promoters.
  • the transgenic mice described herein can be crossed with each other, with a mouse that has increased AD-like pathology, a mouse that is expressing a polypeptide that has a mutation found in a human with AD (e.g., APP, PS-1, or PS-2), or with a reporter mouse.
  • any of the assays of abnormal activity of the EP can be performed to monitor the effectiveness of a candidate compound in decreasing the abnormal activity of the EP.
  • the candidate compound can be directly administered to the mouse.
  • cells from the mice such as fibroblasts, endothelial cells, or neurons, can be assayed in vitro.
  • a transgenic mouse As one example of such a transgenic mouse, we have constructed a mouse overexpressing MPR46 within neurons under the Thy- 1.1 promoter. Following DNA microinjection and implantation of approximately 75 single cell embryos, 29 pups were raised to weaning. From these, two mice were identified that contained the Thy-1.1 MPR46 transgene construct. Both of these founders were shown to transmit the Thy- 1.1 : MPR46 transgene to FI offspring. Fig. 7 A identifies 2 of 11 FI offspring of one such founder as carrying the Thy- 1.1: MPR46 transgene. Brain A ⁇ levels were compared between one such FI Thy-1.1 : MPR46 transgenic mouse and a non-transgenic litter mate (Fig. 7B).
  • Thy-1.1 MPR46 transgenic mouse methods
  • Thy-1.1 promoter which has been used successfully in mice transgenic for mutant APP that show amyloid plaque deposition (Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94:13287-13292, 1997).
  • the Thy-1.1 promoter is valuable in that high levels of transgene expression have been obtained and that expression is restricted within the CNS to neurons.
  • An 8.2 kb EcoRI mouse genomic fragment containing the whole Thy- 1.1 gene forms the core of the transgene constructs (Chang et al., Proc. Natl. Acad. Sci.
  • plasmid was digested with the appropriate restriction enzymes to remove vector sequences, isolated by agarose-gel electrophoresis, and further purified by dialysis in ultra-pure water. Single cell embryos obtained from C57BL6 x CBA/2 FI hybrids were used for injection. Founder transgenic mice were identified and transgene copy number estimated by Southern blot analysis of restriction digested tail DNA using hybridization to a fragment unique to the transgene. Progeny were screened by PCR using human-specific primers. Some FI progeny from the two transmitting founders were examined at 4 weeks of age.
  • Example 5 fibroblasts from DS mice exhibit abnormal activity of the endocytic pathway
  • mice with segmental trisomy 16 (Ts65Dn), an established in vivo model of human "translocation" DS (Holtzman et al., Proc. Natl. Acad. Sci. USA 93:13333-13338, 1996), we found that, at two months of age and prior to the appearance of any neuropathological alterations, many neurons in the neocortex and basal forebrain contained enlarged early endosomes similar to those seen in AD and DS brain (Fig. 8). At six months of age, the endosomal alterations were present within the majority of neurons in the neocortex and basal forebrain and in a substantial number of hippocampal neurons. The abnormal activity of the EP in the Ts65Dn animals is similar to that seen in human SAD and DS.
  • Ts65Dn mice increased neuronal content of APP and the presence of intraneuronal A ⁇ within vesicular compartments (Fig. 9).
  • An antibody directed to the 17-24 region of the A ⁇ peptide was used to probe brain tissue from the Ts65Dn mouse (Fig. 9B), as well as postmortem brain tissue from the neocortex of cases of human fetal, infant, and young DS, and cases obtained from adult patients in the very early stages of AD.
  • Ts65Dn mice Other similarities among the Ts65Dn mice, early DS, and early stage AD were observed. These included abnormalities in proteins associated with the regulation of the activity of the EP.
  • the Ts65Dn mouse can serve as a model for early stages of cellular pathology and A ⁇ formation seen in sporadic AD and DS that are not modeled by existing transgenic mouse models based on mutations associated with familial forms of AD in the presenilin genes and APP gene. This model is useful for assaying candidate compounds for their ability to decrease the foregoing abnormalities and to decrease intracellular production of A ⁇ . Additionally, standard mouse learning and behavior paradigms known to those skilled in the art can be used to assess brain function in Ts65Dn mice administered candidate compounds. The Ts65Dn mouse provides other uses as an in vivo model of the EP upregulation seen in AD and DS.
  • mice that are only partially trisomic for the human homolog of chromosome 21 we can gather additional information on the specificity of the endosomal system upregulation and begin to dissect the genes that may play an essential role.
  • Crossing Ts65Dn mice with a mouse carrying a balanced translocation of chromosome 16, T(12;16)lCje (Huang et al., Arch. Biochem. Biophys. 344:424-432, 1997) generates four genotypes: litter-matched normal control mice; Ts65Dn; the previously described segmental trisomy TslCje (Sago et al., Proc. Natl. Acad. Sci.
  • the T(12;16)lCje mice are phenotypically no ⁇ nal because the translocation is balanced.
  • the TslCje mouse is trisomic for the region of mouse chromosome 16 from the Sodl to Mxl genes, although the Sodl gene is inactive (Sago et al., Proc. Natl. Acad. Sci. USA 95:6256- 6261, 1998). These mice do not have an extra copy of the App gene.
  • TslCje mice have less severe learning deficits than do the Ts65Dn mice and, unlike the Ts65Dn mice, do not show degeneration of basal forebrain cholinergic neurons (BFCN) (Sago et al., Proc. Natl. Acad. Sci. USA 95:6256-6261, 1998).
  • the MslTs65 mice are trisomic for the region of mouse chromosome 16 from the App gene to, but not including, the Sodl gene. Developmental retardation and BFCN degeneration have not been characterized in the MslTs65 mouse.
  • Ts65Dn mice but not TslCje mice, show abnormally large endosomes, this indicates that genes in this part of the chromosome (the App to Sodl region) are essential to the development of this defect.
  • the TslCje mice do not show BFCN degeneration, this result would suggest that endosomal system upregulation and BFCN degeneration share a common genetic underpinning in these animals. While the App gene in this region is likely to be of importance, we suspect that a dosage-effect for APP will not fully explain the Ts65Dn endosomal system upregulation, as we have not seen abnormally large endosomes in the APPswe transgenic mice nor do APP overexpressing mice develop BFCN degeneration. Characterizing the MslTs65 mice that carry an extra copy of App but lack many of the other genes over represented in Ts65Dn mice will directly complement our analysis of the other mice.
  • infusion pumps are implanted in animals at 8 weeks of age and the animals are sacrificed 28 days later.
  • the activity and/or expression of a specific cathepsin or groups of cathepsins is modified by chronic intraventricular infusion of appropriate inhibitors.
  • proteases are inhibited in vivo in the mouse brain using an ALZET osmotic pump brain infusion kit in conjunction with model 2004 mini-osmotic pumps (0.25 mL/hr delivery rate, 28 days) (Frautschy et al., J. Neurosci. 18:8311-8321, 1998).
  • leupeptin 0.5 mmol to 4 mmol
  • leupeptin is a broad-spectrum inhibitor of thiol proteases (e.g., Cat B, Cat L, Cat S)
  • pepstatin 0.1 mmol to 1 mmol
  • Cat D a broad-spectrum inhibitor of thiol proteases
  • M-PAD a broad-spectrum inhibitor of thiol proteases
  • Z-PAD (10 mmol to 100 mmol) inhibits thiol proteases but with greater specificity than leupeptin for Cat B and Cat L.
  • Aprotinin (1 mmol to 30 mmol) broadly inhibits serine proteases and is principally used as a control since the major proteases in lysosomes are not of the serine type.
  • inhibitors are also useful for inducing lysosomal dysfunction, including, for example, vinyl sulfone inhibitors that are selective for cysteine cathepsins and have poor affinity for calpains, and peptidyl fluoromethylketone inhibitors that preferentially inhibit Cat B (Palmer et al., J. Med. Chem. 38:3193-3196, 1995; Ahmed et al., Biochem. Pharmacol. 44: 1201-1207, 1992; Esser et al., Arthritis Rheum 37:236-247, 1994; Riese et al., J. Clin. Invest. 101:2351-2363, 1998).
  • mice To administer the candidate compounds in the presence or absence of inhibitor(s), eight week old mice are anesthetized with pentobarbital (40-50 mg/kg i.p.) and placed in a stereotaxic apparatus with mouse adapter (David Kopf Instruments, Tujunga, CA). After sacrifice, placement of the probe into the lateral ventricle is confirmed and one hemi-brain immersion- fixed and the other hemi-brain homogenized for protease assays.
  • We determine the efficacy of the candidate compound by assaying the activity of proteases in brain homogenates (e.g., Cat B, Cat L, Cat D, and trypsin (Brank et al., Free Radic. Biol. Med.
  • Figs. 11C-F increased brain levels of Cat D, a protease not directly affected by leupeptin, is shown by immunohistochemistry (Fig. 1 ID), Western blot analysis (Fig.
  • Abnormal tau conformation and hyperphosphorylated tau is examined using suitable monoclonal antibodies (e.g., MCI (Jicha et al., J. Neurosci. Res. 48:128-132) and AT8 (Innogenetics, Gent, Belgium)).
  • EM analysis can be performed to characterize cytoskeletal changes in neurons (Nixon, Bioessays 20:798-807, 1998).
  • the magnitude of neurodegeneration in the mice can be determined by Nissl stain.
  • a ⁇ levels can be determined in mice administered inhibitors with or without candidate compounds.
  • Levels of APP, APPs, APP carboxy- te ⁇ ninal fragments, and A ⁇ production can be examined using methods described herein. Additionally, the presence of A ⁇ in endosomal and lysosomal compartments is assayed by double-label immunocytochemistry using antibodies to A ⁇ and appropriate markers for these compartments (e.g., rab5).
  • PS l M146L /APPswe transgenic mice As a second example of the usefulness of inducing lysosomal system dysfunction by protease inhibitor infusion, we treated PS l M146L /APPswe transgenic mice, an established model of AD-like pathology (Duff et al., Nature 383:710-713, 1996). Infusion pumps containing 5 mg/mL leupeptin were implanted into PSl M146L /APPswe transgenic mice at 1 year of age. Animals were sacrificed 4 weeks later. Nissl staining of a PS M146L /APPswe mouse infused with leupeptin showed substantial cortical neuronal hypotrophy, chromatolysis and possibly loss of neurons (Figs.
  • mice were sacrificed and fixed tissue examined for lipofuscin autofluorescence, an indication of reduced lysosomal hydrolysis and accumulation of nondegraded material. There was an increase in lipofuscin autofluorescence in leupeptin- treated mice (Fig. 9B) when compared to animals receiving vehicle (20 mM HEPES) alone (Fig. 9A). Additionally, Cat D, a protease not directly affected by leupeptin, was examined in these mice. Immunolabeling of tissue sections with antibodies specific for Cat D showed a substantial increase in Cat D expression within neurons of leupeptin-treated animals (Figs.
  • PSl M146L /APPswe transgenic mice PSl M146L /APPswe transgenic mice.
  • Infusion pumps containing either 2 mg/mL pepstatin or 10 mg/mL leupeptin were implanted into PSl M146L /APPswe transgenic mice at 8 weeks of age. Animals were sacrificed at 12 weeks of age, and total A ⁇ levels were determined by ELISA following formic acid extraction (Fig. 10A). Total A ⁇ levels in the pepstatin-treated mice were found to be reduced to half the level detected in untreated 12 week old PSl M146L /APPswe transgenic mice. Leupeptin treatment, on the other hand, increased total A ⁇ by 1.8 times when compared to untreated mice.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • the compounds, compositions, or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline.
  • Treatment may be accomplished directly, e.g., by treating the animal with antagonists which disrupt, suppress, attenuate, or neutralize the biological events associated with AD.
  • Preferable routes of administration include, for example, inhalation or subcutaneous, intravenous, interperitoneally, intramuscular, intraventricular infusion, or intradermal injections which provide continuous, sustained levels of the drug in the patient.
  • Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a compound in a physiologically-acceptable carrier.
  • Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin.
  • the amount of the compound to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease and extensiveness of the disease.
  • a compound is administered at a dosage that decreases activity of the endocytic pathway.
  • a compound is administered typically in the range of O.l ng - 10 g/kg body weight.
  • candidate compounds can be assayed for their ability to reduce the alterations in endocytosis and hydrolase trafficking to early endosomes observed in human brain. Additionally, candidate compounds can be assayed for their ability to decrease A ⁇ production. These assays are described in more detail below.
  • ⁇ Receptor mediated uptake is assayed by binding fluorescent-labeled transferrin (Cy3-transferrin, 25 ⁇ g/ml) at 4°C for 1 hr to living cells followed by warming to 37°C for periods of 0, 3, 6, 8, and 20 min. Cells fixed after 6 min, which we have found to have maximum uptake into early endosomes, are quantitated for the number of Cy3-positive endosomes. In addition, double labeling experiments are performed to show the coincidence of Cy 3 -transferrin and rab5. In these assays, it is preferred that transformed cells are compared to parental cells.
  • Double-labeling with the specific antibodies (rab5 and Cat D or Cat B) and confocal microscopy are used to show coincidence of lysosomal hydrolases and early endosomes.
  • Uptake of BODIPY-pepstatin, which specifically binds to enzymatically active Cat D, is a useful method to detect increased Cat D levels in early endosomes. 1 mM BODIPY-pepstatin is added to the growth medium, and cells washed at various time-points (10 min to 5 hr), fixed, and labeled with antibodies specific for early endosomes and examined as above.
  • the specificity of increased BODIPY-pepstatin uptake is determined by including an excess of unlabeled pepstatin in the medium (20 mM).
  • Cat D and Cat B are immunoprecipitated from cell lysates and from the growth medium using specific antibodies.
  • 5 mM Man-6-P is added to the chase medium to promote secretion of hydrolases delivered to the cell surface/early endosomes into the medium (Watanabe et al., Proc. Natl. Acad. Sci. USA 87:8036-8040, 1990).
  • a mouse from a transgenic line that is administered a candidate compound is analyzed to determine whether it exhibits evidence of decreased endocytosis or less misfrafficking of hydrolases to early endosomes compared to a transgenic mouse from the same line but not given a candidate compound. This can be performed using any of the assays described herein. In one example, sections of mouse brain are assessed by labeling with antibodies to rab5, Cat D, and MPR46. Whether endocytosis is decreased in the transgenic mice administered the compound can be determined using semiquantitative densitometry.
  • transgenic mice described herein can be crossed with each other, with a mouse that has increased AD-like pathology, a mouse that is expressing a polypeptide that has a mutation found in human with AD (e.g., APP, PS-1, or PS-2), or with a reporter mouse.
  • Transgenic mice from these crosses that have been administered a candidate compound are examined, for example, at 2, 5.5, or 12 months of age.
  • the A ⁇ sandwich ELISA is generally known to those skilled in the art, with both A ⁇ ELISA kits (Biosource International, Camavillo, CA) and appropriate antibodies (e.g., 4G8, GE10; Seneteck, PLC, Napa, CA) available commercially.
  • a ⁇ sandwich ELISA Nunc-Immuno Plates (Nunc A/S Roskilde, Denmark) were coated overnight at 4% using antibodies specific for A ⁇ 40 or A ⁇ 42 in 100 mM bicarbonate buffer, pH 9.6. Remaining protein binding sites were blocked by incubating with 1% Block Ace (Yukijirushi Milk, Sapporo Japan) in PBS for 4 hours at room temperature.
  • 10% (w/v) homogenates were prepared from a hemibrain in 20mM Tris, 250 mM sucrose, ImM EDTA, lmM EGTA, protease inhibitors, pH 7.4, and stored frozen at - 70°C.
  • 1ml of the brain homogenate was extracted in diethylamine (Sigma, St. Louis, MI) by adding an equal volume of 0.4% DEA in lOOmM NaCl, re-homogenized, and centrifuged for 1 hour at 100,000 x g.
  • the supernatant was collected, neutralized with 0.1 volume 0.5 M Tris, pH 6.8, and loaded in duplicate wells both neat and diluted 1 :2 in EC buffer (20mM Na phosphate, 2 mM EDTA, 400mM NaCl, 0.2 BSA, 0.4% Block Ace, 0.95% CHAPS).
  • the DEA extraction protocol has been shown to efficiently recover immunoreactive A ⁇ from mouse brain homogenates and leave both full-length and sAPP in the 100,000 x g pellet (Savage et al., J. Neurosci. 18:1743-1752, 1998).
  • conditioned media collected from cells was loaded neat and 1 :2.
  • a ⁇ -40 and A ⁇ -42 peptide standards were purchased from American Peptide Co.
  • ELISA plates were incubated overnight at 4°C with samples and standards. A ⁇ was detected by incubating for 4 hours at room temperature with an HPR-conjugated anti-A ⁇ antibody in 20mM Na phosphate, 2mM EDTA, 400mM NaCl, 1.0% BSA. ELISA plates were developed using a color reaction (ABTS Peroxidase Substrate System, Kirkegaard & Perry, Gaithersburg, MD) and the OD 450 read.
  • ABTS Peroxidase Substrate System Kirkegaard & Perry, Gaithersburg, MD
  • L-cells andN2a cells are maintained at 37°C and 5% C0 2 in high glucose DMEM supplemented with 10% FBS, 2mM glutamax I, and penicillin/streptomycin.
  • medium is supplemented with 400 ⁇ g/ml of G418, 200 ⁇ g/ml of hygromycin B, and/or HAT as is appropriate.
  • IMR-32 neuroblastoma cells are maintained in 60% DMEM, 30% Ham's-F12, 5% ⁇ MEM, 1% FCS, 4% NCS, 0.6% glucose, 200mM glutamine, and 15mM HEPES. Neuronal differentiation is achieved by supplementing medium with 1 mM dibutyryl cAMP and 2.5 ⁇ M 5-BDU.
  • Transfected cells are incubated in 20mM butyrate in growth medium for 24-48 hr prior to fixation to induce expression of transfected cDNA and fixed in 4% PFA, 5% sucrose in PBS (pH 7.3) at RT for 20-35 min, rinsed, and labeled with specific antibody in PBS containing 10% serum with 0.1% saponin. Coverslips are washed after primary antibody binding and secondary antibody is bound for 2 hr at RT. The coverslips are then be rinsed, mounted and examined by immunofluorescence.
  • Approximately 5 x 10 5 cells are seeded in 35 mm dishes for 16 hr, followed by neuronal differentiation and/or induction of expression of transfected cDNA.
  • Cells are incubated in methionine- and cysteine-free DMEM for 20 min before a 15-minute incubation with 100-200 ⁇ Ci/mL TRANS 35 S-LABEL (Dupont-NEN), or for 4 hr with 500 ⁇ Ci/ml for A ⁇ IP.
  • DMEM 10% FBS and 2mM methionine at 4°C cells are lysed immediately or incubated with 2 mM unlabeled methionine in DMEM with 10% FBS for chase periods of 15 min to 8 hr.
  • APP and APP metabolites are immunoprecipitated using any of a number of antibodies known to those skilled in the art, followed by separation using SDS-PAGE. Gels are exposed to X-ray film or are analyzed quantitatively using a Phosphoimager. Immunocytochemistry and digital confocal microscopic analysis
  • Sections of fixed transgenic mouse brain or fixed cultured cells are processed as previously described (Cataldo et al., Neuron 14: 671-680, 1995; Cataldo et al., J. Neuropathol. Exp. Neurol. 55: 704-715, 1996; Cataldo et al., Adv. Exp. Med. Biol. 389: 271-280, 1996; Cataldo et al., J. Neurosci. 16: 186- 199, 1996; Cataldo et al., Proc. Natl. Acad. Sci. USA 87: 3861-3865, 1990; Cataldo et al., Brain Res.
  • Ts65Dn mice made transgenic for human APOE ⁇ 2 and ⁇ 4, as well as other transgenic mouse models described herein, are studied initially by immunocytochemical analysis with rab5, rabaptin 5 and EEA 1 to . determine if the APOE genotype accentuates morphological changes in early endosomes. The effect of ApoE on exacerbating EP changes is further examined by crossing APOE allele-specific mice with Ts65Dn mice.
  • a compound that decreases the activity of the EP can be further tested for AD-like abnormalities in physiology, anatomy, or behavior using assays known to those skilled in the art, including those described in U.S. patent 5,877,399, hereby incorporated by reference.

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Abstract

Cette invention se rapporte à des procédés permettant d'identifier des composés utiles au traitement de la maladie d'Alzheimer.
PCT/US2000/011401 1999-04-30 2000-04-28 Procedes d'identification de composes pour le traitement de la maladie d'alzheimer Ceased WO2000067016A1 (fr)

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